Modular nanodevices for smart adaptable vaccines

ABSTRACT

Modular nanoparticle vaccine compositions and methods of making and using the same have been developed. Modular nanoparticle vaccine compositions comprise an antigen encapsulated in a polymeric particle and adaptor elements which modularly couple functional elements to the particle. The modular design of these vaccine compositions, which involves flexible addition and subtraction of antigen, adjuvant, immune potentiators, molecular recognition and transport mediation elements, as well as intracellular uptake mediators, allows for exquisite control over variables that are important in optimizing an effective vaccine delivery system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. Ser. No. 12/527,143,filed Aug. 13, 2009, under 35 U.S.C. §371 of PCT/US2008/054086 filed onFeb. 15, 2008, which claims priority to and benefit of U.S. ProvisionalPatent Application No. 60/890,133 filed on Feb. 15, 2007 and U.S.Provisional Patent Application No. 61/000,016 filed on Oct. 23, 2007,and where permissible is incorporated by reference in its entirety.

GOVERNMENT SUPPORT

The United States government has certain rights in this invention byvirtue of National Science Foundation Grant Number 0609326 to TarekFahmy.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted on Aug. 8, 2014 as a text file named“YU_(—)4549 CIP_ST25.txt,” created on Dec. 1, 2014, and having a size of8,952 bytes is hereby incorporated by reference pursuant to 37 C.F.R.§1.52(e)(5).

FIELD OF THE INVENTION

The present disclosure generally relates to the field of modularnano-scale vaccine compositions and methods of making and using thesecompositions.

BACKGROUND OF THE INVENTION

Preventative vaccines have eliminated smallpox and nearly eliminatedpolio, two of the worst global infectious diseases. By contrast vaccinesfor many other infectious diseases, such as malaria and HIV, whichinvolve intracellular pathogens, are poorly developed or simplyunavailable (Singh and O'Hagan, Pharm. Res., 19(6):715-28 (2002);O'Hagan and Valiante, Nat. Rev. Drug Discov., 2(9):727-35 (2003)). Thelack of such vaccines results in two million unnecessary deaths eachyear in many parts of the world (WHO, State of the Art New Vaccines(2003)).

Several key variables are needed in the design of effective vaccines(Pashine, et al., Nat. Med., 11(4 Suppl):563-8 (2005), Bramwell andPerrie, Drug Discovery Today, 10(22):1527-34 (2005)). The first variableis the form of the antigen itself, which can be whole inactivated orattenuated organisms, purified proteins and peptides, or DNA encodedantigens. Human pathogens are continually emerging and changing (e.g.SARS, avian flu) meaning that new potential immunogens are constantlyappearing. Thus, there is a clear need to design vaccine systems thatcan rapidly and efficiently test the efficacy of vaccines involving newantigens (Bramwell, et al., Adv. Drug Deliv Rev. 57(9):1247-65 (2005)).Large scale and safe production of stable vaccine products typicallyinvolves the purification of natural or recombinant forms of antigenicsubunits. Once purified, however, individual antigens often become lessimmunogenic compared to whole pathogens or crude extracts, necessitatinga means to amplify the immune response against the purified subunitantigen. Thus, a second necessary component of a vaccine involvesproviding an adjuvant or other means for potentiating or stimulatingboth the innate and adaptive arms of the immune system to the antigensubunit (Pashine, et al., Nat. Med., 11(4 Suppl):563-8 (2005), Bramwelland Perrie, Drug Discovery Today, 10(22):1527-34 (2005)).

Immune potentiators may include bacterial products, toxins or othermolecules that augment specific immunity. Potentiators have variousbenefits, but also attendant risks such as triggering deleteriousinflammatory responses. To affect optimal stimulation to a givenantigen, a formulation is needed that delivers the correct amount ofantigen in a repetitive or sustained fashion, to the appropriate immunecells and to the appropriate compartments within those cells. Thus, adesigned delivery vehicle (adjuvant) should target the vaccine antigenand facilitate delivery of both antigen and immune potentiatingmolecules selectively to target cells of the immune system. This ishighly reminiscent of the strategy taken by viruses that inactivatespecific components of the immune system during infection. Traditionalmethods for increasing the effectiveness of vaccines have focused onco-administration of adjuvants or use of a delivery system.

While the adjuvant role is critical, there are obvious risks, costs andlimitations associated with this traditional approach. For example,currently available adjuvants, represented predominately by colloidalalum (aluminum sulfate or aluminum hydroxide) or montanide polymers,have a limited capacity to adsorb many antigens and have greatly limitedimmunostimulatory properties (Gupta and Siber, Vaccine, 13(14):1263-76(1995); Lindblad, Vaccine, 22(27-28):3658-68 (2004)). There are alsorisks associated with using live attenuated vaccines and allergic sideeffects associated with aluminum salts (Lindblad, Vaccine,22(27-28):3658-68 (2004); Gupta, et al., Vaccine, 11(3):293-306 (1993)).Additionally, because of the historical emphasis on eliciting humoralimmune responses, most adjuvants are optimized for effective inductionof high antibody serum titers, but are ineffective at eliciting a strongcellular, T cell-mediated immune response or strong mucosal immuneresponse. T cell responses are essential for inducing lasting viralimmunity (or immune responses to cancer); mucosal immunity is essentialfor protective responses to cellular and viral pathogens that aretransmitted through mucosal surfaces (e.g. human immunodeficiency virus,HIV; herpes simplex virus, HSV; enteric pathogens). These factors,coupled with the difficulties of manufacture, storage, and transporthave together greatly limited the utility of current approaches in theclinic and in the field (O'Hagan and Valiante, Nat. Rev. Drug Discov.,2(9):727-35 (2003); Sigh and Srivastava, Curr. HIV Res., 1(3):309-20(2003), Singh and O'Hagan, Nat. Biotechnol., 17(11):1075-81 (1999)).

Thus, in addition to economic factors, as outlined above, there are anumber of significant scientific challenges that have limited thedevelopment of vaccines for deadly diseases. First, few if anyapproaches are available that efficiently prime cell-mediated immunityby direct intracellular delivery of an antigen. Second, ‘tunable’adjuvants, that is, adjuvants that can be engineered to optimize themagnitude and direction of an immune response (Jiang, et al., Adv. DrugDeliv. Rev., 57(3):391-410 (2005); Sesardic and Dobbelaer, Vaccine,22(19):2452-6 (2004)) have not been developed. Third, alternatives arenot available for the general requirement for parenteral (i.e.subcutaneous or intramuscular injection) administration of vaccines, asituation that has made it difficult to deploy vaccines inunderdeveloped countries where medical support systems, resources, andcold-storage are limited. Finally, there is no general approach todesigning oral vaccines targeted to both systemic and mucosal immunity.This would be highly advantageous since oral vaccines are significantlyless expensive to administer and transport. Thus, there is a criticalneed for safe and stable vaccine systems that would address all thesefactors (Friede and Aguado, Adv. Drug Deliv. Rev., 57(3):325-31 (2005);Storni, et al., Adv. Drug Deliv. Rev., 57(3):333-55 (2005); Gupta, etal., Adv. Drug Deliv. Rev., 32(3):225-246 (1998); Aguado and Lambert,Immunobiology, 184(2-3):113-25 (1992)).

It is therefore an object of the invention to provide stable vaccineformulations which can be orally administered.

It is another object of the invention to provide modular nanoparticulatevaccine compositions which provide for flexible addition and subtractionof elements.

It is still another object of the invention to provide means formodulating an immune response, either to increase or decrease theresponse, or bias the response to a humoral or cellular immune response.

It is a further object of the invention to provide methods for makingand using such modular nanoparticulate vaccine compositions.

SUMMARY OF THE INVENTION

Modular nanoparticle vaccine compositions and methods of making andusing them have been developed. The modular design of these nanoparticlevaccine compositions, which involves flexible addition and subtractionof antigen, adjuvant and/or immune potentiators, molecular recognitionfactors, and transport mediation elements, as well as intracellularuptake mediators, allows for exquisite control over many of thevariables that are important for optimizing an effective vaccinedelivery system. A key feature of these nanodevices is their ability tobe selectively targeted to those cells of the immune system that aremost closely associated with producing the desired immunologicalresponse for a given vaccine. This is accomplished by encapsulating anyvaccine antigen within the nanoparticles, together with the activatorsof the desired immune activity. The nanoparticle surface is thenmodified by the direct or indirect coupling of targeting molecules, suchas antibodies, that guide the entire nanodevice to specific cell types(such as dendritic cells) associated with stimulating or suppressingimmune responses. The targeted particles are constructed to bind to theintended cell type, to be internalized by endocytosis, and then todissociate, thereby releasing the encapsulated antigen and immuneactivators (adjuvants). The modular nature of the nanodevice enablesrapid production and the ability to modify the nanoparticle surface withany of a variety of targeting molecules, enabling targeting to differentcell types, such as various dendritic cell subsets, epithelial cells, ormacrophages. The adjuvant composition can also be easily altered toenable the systematic assessment of optimal targeting and compositionfor any desired application. The nanodevices can be easily characterizedbiochemically using conventional ELISA and flow cytometry assays, and byin vitro or in vivo assays for antigen presentation and immunestimulation.

Modular nanoparticle vaccine compositions include an antigenincorporated or encapsulated in a polymeric nanoparticle. Antigens maybe viral, bacterial, parasitic, allergen, toxoid, tumor-specific ortumor-associated antigens, which can be one or more proteins,carbohydrates, lipids, nucleic acids, or combinations thereof. Thenanoparticle further includes adaptor elements which modularly couplefunctional elements to the particle. In the preferred embodiment, theadaptor elements are fatty acids, hydrophobic or amphipathic peptides,or hydrophobic polymers. Adaptor elements can be conjugated to affinitytags, which allow for modular assembly and disassembly of functionalelements which are conjugated to complementary affinity tags to thenanoparticle. Functional elements impart useful functions to thenanoparticle compositions. Functional elements may include, for example,dendritic cell targeting molecules, epithelial cell targeting molecules,pH-sensitive or non-pH-sensitive molecules which protect the vaccinecomposition from hydrolysis and degradation in low pH environments, andendosome-disrupting agents. Nanoparticle vaccine compositions mayfurther include adjuvants, contrast agents and other markers andpharmaceutically acceptable excipients.

The ability to target exogenous antigens to internalizing surfacemolecules on antigen-presenting cells facilitates the uptake of antigensand their presentation to lymphocytes and thus overcomes a majorrate-limiting step in vaccination. The ability to target vaccinecompositions to epithelial cells in the digestive tract greatlyfacilitates the ability of a vaccine to induce mucosal and systemicimmunity when administered orally. Molecules which protect the vaccinecomposition from hydrolysis and degradation in low pH environments alsoenhance the efficacy of vaccines administered orally.Endosome-disrupting agents function to cause limited disruption ofendosome-lysosome membranes during antigen uptake by antigen-presentingcells. This allows the antigen to enter the cytoplasm and be presentedon MHC class I molecules on the surface of antigen-presenting cells in aprocess known as cross-presentation. Cross-presentation allows for theactivation of cytotoxic CD8 positive T cells which greatly enhances theeffectiveness of vaccination. The modular nanoparticulate vaccinecompositions offer several advantages over other vaccines: 1) targetingof different cells, thereby enabling optimal selection of differenttissue and priming for antigen presentation; 2) delivery of a widevariety of antigens of clinical importance; and 3) rapid assembly ofdifferent combinations of protective, recognition and antigen modules toaffect a broad-spectrum potent vaccine response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph demonstrating inhibition of CD3-stimulated T-cellproliferation (number of cells) when T-cells are exposed todoxorubicin-loaded particles modified with an antibody (-▪-) thatrecognizes T-cells at the indicated concentration (mg/ml). Controls aredoxorubicin-loaded nanoparticles without antibody (-□-) and blanknanoparticles (-▴-).

FIG. 2A is a graph showing that spleen cells obtained from mice threedays after subcutaneous immunization with ovalbumin-encapsulated,LPS-modified, nanoparticles (--) proliferated (number of cells×10⁶) inresponse to immobilized ovalbumin, thus demonstrating memory to theantigen. Controls are immobilized antigen (-∘-) and blank nanoparticles(-♦-).

FIG. 2B is a graph showing that spleen cells obtained from micefollowing oral immunization with ovalbumin-encapsulated, LPS-modified,nanoparticles (--) proliferated (absorbance) in response to immobilizedovalbumin, demonstrating the efficacy of the particles in inducingimmunity through oral administration. Controls are immobilized (-∘-)antigen and blank nanoparticles (-♦-).

FIG. 3 is a graph showing release of IL-2 (ng/ml) by CD8 (OT-1) (--)and CD4 (OT-II) (-▪-) positive T-cells as a function of theconcentration of Endoporter (μl/ml) incubated with mouse bonemarrow-derived dendritic cells. This graph demonstrates that inclusionof increasing concentrations of Endoporter enhanced cross presentationof antigen to MHC class I-restricted CD8 T-cells, while presentation toMHC class II-restricted CD4 T-cells was not diminished.

FIG. 4 is a graph demonstrating that nanoparticles conjugated to elastin(--) retain incorporated drug (mg) at low pH (pH=2) over time morereadily than non-conjugated particles (-▪-).

FIG. 5 is a diagram showing the mechanism of endosomal disruptionfollowing uptake of cargo by cells using endoporter as the exemplaryendosomal targeting agent.

FIG. 6 is a bar graph showing endosomal disruption as percentage (%) ofbone marrow derived dendritic cells positive for 26.D16-PE whenincubated for 24 hours with PLGA or liposomal nanoparticlesencapsulating a positively charged macromolecule dendrimer (G5) and theantigen ovalbumin.

FIG. 7 is a dot plot showing endosomal disruption as percentage (%) ofbone marrow derived macrophages positive for 26.D16-PE when incubatedfor 24 hours with PLGA-PEG or liposomal nanoparticles encapsulating apositively charged macromolecule dendrimer and the antigen ovalbumin.

FIG. 8 is a bar graph showing percentage (%) of dendritic cells withdisrupted endosomes when the cells are incubated with PLGA-dendrimer(G4) or PLGA-cyclodextrin nanoparticles without (1) or with (2) carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP).

FIG. 9 is a bar graph showing percentage (%) of green fluorescentprotein (GFP)-positive bone marrow derived dendritic cells transfectedwith monophosphoryl lipid A (MPLA) or liposomal nanoparticlesco-encapsulating GFP, the dendrimer G5 and/or CpG DNA. LED stands for“Lipid Encapsulated Dendrimer”. The different groups being: −/G5 LED isa Lipid Encapsulating Dendrimer of Generation 5 with no surfacemodification; (−/G5+CpG) LED is the same with co-encapsulated CpGassociated with the encapsulated dendrimer; MPLA/G5 LED is the same withMPLA surface modified lipid and encapsulating G5 Dendrimer;MPLA/(G5+CpG) LED is MPLA surface modified lipid encapsulating CpGassociated with the G5 Dendrimer; MPLA/G5 LED & −/(G5+CpG) LED are twoparticles with and without MPLA and unmodified particle encapsulatingCpG; Lipofectamine is a gold standard for gene transfection.

FIG. 10 is a bar graph showing change in MFI targeted/non targetedantigen presenting cells (APCs) when BDCA3+ and DC SIGN+DC subsets aretargeted with nanoparticles surface modified with anti-BDCA3 andanti-DC-SIGN antibodies, respectively.

FIGS. 11A-11D are bar graphs showing mean cytokine expression levels(IL-15, IFN-λ and TNF-α in pg/ml, and IL-6 and IL-8 in pg/dl, ±SEM) per30,000 APCs for BDCA3+ or DC-SIGN+DC subsets obtained from threedifferent healthy donors.

FIG. 12 is a bar graph showing mean percentage (%, ±SEM) of CD8+ cellsproducing IFN-γ when stimulated by Mo-DCs or BDCA3+ MDCs loaded witheither blank NP or NP-CEF.

DETAILED DESCRIPTION OF THE INVENTION

A solution to the vaccine problem requires a systematic approach thataddresses each of the design challenges discussed above. Viruses andpathogens that elicit or subvert immune responses are, in essence, smallparticles endowed with the ability to interact with or avoid cells ofthe immune system in a variety of ways. The vaccines described hereinare based on an approach in which nanoscale modules are assembled intounits that are optimized for stimulating immune responses to a specificpathogen. The principles of nanoassembly is used to design safe vaccinevectors that are highly optimized to protect against disease and providenew treatment options for disorders such as asthma, allergy, and cancer.

I. DEFINITIONS

“Affinity tags” are defined herein as molecular species which formhighly specific, non-covalent, physiochemical interactions with definedbinding partners. Affinity tags which form highly specific,non-covalent, physiochemical interactions with one another are definedherein as “complementary”.

“Adaptor elements” are defined herein as molecular entities whichassociate with polymeric nanoparticles and provide substrates thatfacilitate the modular assembly and disassembly of functional elementsonto the nanoparticle. Adaptor elements can be conjugated to affinitytags. Affinity tags allow for flexible assembly and disassembly offunctional elements which are conjugated to affinity tags that formhighly specific, noncovalent, physiochemical interactions with affinitytags conjugated to adaptor elements. Adaptor elements can also becovalently coupled to functional elements in the absence of affinitytags.

“Functional elements” are defined herein as molecular entities whichassociate with nanoparticles and impart a particular function to thenanoparticle. Functional elements can associate with nanoparticlesthrough adaptor elements, or through direct association with thenanoparticle surface. Functional elements can be conjugated to affinitytags which form highly specific, noncovalent, physiochemicalinteractions with complementary affinity tags conjugated to adaptorelements. Thus, functional elements can be coupled to adaptor elementsnoncovalently through affinity tags. Alternatively, functional elementscan be covalently coupled to adaptor elements in the absence of affinitytags. Functional elements can also be covalently or noncovalentlyassociated with the surface of nanoparticles without the use of adaptorelements.

An “antigen” is defined herein as a molecule which contains one or moreepitopes that will stimulate a host's immune system to make a cellularantigen-specific immune response, and/or a humoral antibody response.Antigens can be peptides, proteins, polysaccharides, saccharides,lipids, nucleic acids, and combinations thereof. The antigen can bederived from a virus, bacterium, parasite, plant, protozoan, fungus,tissue or transformed cell such as a cancer or leukemic cell and can bea whole cell or immunogenic component thereof, e.g., cell wallcomponents. An antigen may be an oligonucleotide or polynucleotide whichexpresses an antigen. Antigens can be natural or synthetic antigens, forexample, haptens, polyepitopes, flanking epitopes, and other recombinantor synthetically derived antigens (Bergmann, et al., Eur. J. Immunol.,23:2777-2781 (1993); Bergmann, et al., J. Immunol., 157:3242-3249(1996); Suhrbier, Immunol. and Cell Biol., 75:402-408 (1997).

A “tumor-specific antigen” is defined herein as an antigen that isunique to tumor cells and does not occur in or on other cells in thebody.

A “tumor-associated antigen” is defined herein as an antigen that is notunique to a tumor cell and is also expressed in or on a normal cellunder conditions that fail to induce an immune response to the antigen.

An “adjuvant” is defined herein as a substance increasing the immuneresponse to other antigens when administered with other antigens.Adjuvants are also referred to herein as “immune potentiators” and“immune modulators”.

“Antigen-presenting cells” are defined herein as highly specializedcells that can process antigens and display their peptide fragments onthe cell surface together with molecules required for lymphocyteactivation. The major antigen-presenting cells for T cells are dendriticcells, macrophages and B cells. The major antigen-presenting cells for Bcells are follicular dendritic cells.

“Cross-presentation” is defined herein as the ability ofantigen-presenting cells to take up, process and present extracellularantigens with MHC class I molecules to CD8 T cells (cytotoxic T cells).This process induces cellular immunity against most tumors and againstviruses that do not infect antigen-presenting cells. Cross-presentationis also required for induction of cytotoxic immunity by vaccination withprotein antigens, for example in tumor vaccination.

An “endosome-disrupting agent” is defined herein as any agent whichcauses disruption of endosomal membranes during endocytosis.Endosome-disrupting agents facilitate the transit of extracellularantigens into the cytoplasm of antigen-presenting cells, where they canbe imported into the endoplasmic reticulum and processed forcross-presentation on MHC class I molecules at the cell surface.

“Dendritic cell targeting molecules” are defined herein as moleculesthat target and facilitate endocytosis of nanoparticles by dendriticcells. Dendritic cell targeting molecules may be directly coupled tonanoparticles, or may be coupled to nanoparticles through adaptorelements. In a preferred embodiment the dendritic cell targetingmolecules are functionally coupled to adaptor elements.

“Epithelial cell targeting molecules” are defined herein as moleculesthat target the nanoparticles to epithelium and mediate transcytosis tounderlying antigen-presenting cells. Epithelial cell targeting moleculesmay be directly coupled to nanoparticles, or may be coupled tonanoparticles through adaptor elements. In a preferred embodiment theepithelial cell targeting molecules are functionally coupled to adaptorelements.

As used herein, the phrase that a molecule “specifically binds” to atarget refers to a binding reaction which is determinative of thepresence of the molecule in the presence of a heterogeneous populationof other biologics. Thus, under designated immunoassay conditions, aspecified molecule binds preferentially to a particular target and doesnot bind in a significant amount to other biologics present in thesample. Specific binding of an antibody to a target under suchconditions requires the antibody be selected for its specificity to thetarget. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, New York, for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity. Specific binding between two entities means anaffinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Affinities greaterthan 10⁸ M⁻¹ are preferred.

As used herein, the terms “antibody” or “immunoglobulin” are used toinclude intact antibodies and binding fragments thereof. Typically,fragments compete with the intact antibody from which they were derivedfor specific binding to an antigen fragment including separate heavychains, light chains Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragments areproduced by recombinant DNA techniques, or by enzymatic or chemicalseparation of intact immunoglobulins. The term “antibody” also includesone or more immunoglobulin chains that are chemically conjugated to, orexpressed as, fusion proteins with other proteins. The term “antibody”also includes bispecific antibody. A bispecific or bifunctional antibodyis an artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites. Bispecific antibodies can beproduced by a variety of methods including fusion of hybridomas orlinking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp.Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553(1992).

As used herein, the terms “epitope” or “antigenic determinant” refer toa site on an antigen to which B and/or T cells respond. B-cell epitopescan be formed both from contiguous amino acids or noncontiguous aminoacids juxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5 or 8-10 amino acids, in a uniquespatial conformation. Methods of determining spatial conformation ofepitopes include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance. See, e.g., Epitope Mapping Protocols inMethods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).Antibodies that recognize the same epitope can be identified in a simpleimmunoassay showing the ability of one antibody to block the binding ofanother antibody to a target antigen. T-cells recognize continuousepitopes of about nine amino acids for CD8 cells or about 13-15 aminoacids for CD4 cells. T cells that recognize the epitope can beidentified by in vitro assays that measure antigen-dependentproliferation, as determined by ³H-thymidine incorporation by primed Tcells in response to an epitope (Burke et al., J. Inf. Dis. 170, 1110-19(1994)), by antigen-dependent killing (cytotoxic T lymphocyte assay,Tigges et al., J. Immunol. 156, 3901-3910) or by cytokine secretion.

As used herein, the terms “immunologic”, “immunological” or “immune”response is the development of a humoral (antibody mediated) and/or acellular (mediated by antigen-specific T cells or their secretionproducts) response directed against an antigen. Such a response can bean active response induced by administration of immunogen or a passiveresponse induced by administration of antibody or primed T-cells. Acellular immune response is elicited by the presentation of polypeptideepitopes in association with Class I or Class II MHC molecules toactivate antigen-specific CD4⁺ T helper cells and/or CD8⁺ cytotoxic Tcells. The response may also involve activation of monocytes,macrophages, NK cells, basophils, dendritic cells, astrocytes, microgliacells, eosinophils or other components of innate immunity. The presenceof a cell-mediated immunological response can be determined byproliferation assays (CD4⁺ T cells) or CTL (cytotoxic T lymphocyte)assays. The relative contributions of humoral and cellular responses tothe protective or therapeutic effect of an immunogen can bedistinguished by separately isolating antibodies and T-cells from animmunized syngeneic animal and measuring protective or therapeuticeffect in a second subject.

As used herein, a “costimulatory polypeptide” or a “costimulatorymolecule” is a polypeptide that, upon interaction with a cell-surfacemolecule on T cells, enhances T cell responses, enhances proliferationof T cells, enhances production and/or secretion of cytokines by Tcells, stimulates differentiation and effector functions of T cells orpromotes survival of T cells relative to T cells not contacted with acostimulatory peptide.

The terms “individual”, “host”, “subject”, and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, murines, simians, humans, mammalian farm animals, mammaliansport animals, and mammalian pets.

II. MODULAR NANOPARTICULATE VACCINE COMPOSITIONS

Modular nanodevice vaccine systems are constructed from nanoparticles.The modular design of these nanoparticle vaccine compositions, whichinvolves flexible addition and subtraction of antigen, adjuvant, immunepotentiators, molecular recognition, and/or transport mediationelements, as well as intracellular uptake mediators, allows forexquisite control over many of the variables that are important foroptimizing an effective vaccine delivery system.

A. Polymeric Nanoparticles

As used herein, nanoparticles generally refers to particles in the rangeof between 500 nm to less than 0.5 nm, preferably having a diameter thatis between 50 and 500 nm.

The polymer that forms the core of the modular vaccine nanoparticle maybe any biodegradable or non-biodegradable synthetic or natural polymer.In a preferred embodiment, the polymer is a biodegradable polymer. Thesesystems have several features that make them ideal materials for thefabrication of a vaccine nanodevice: 1) control over the size range offabrication, down to 100 nm or less, an important feature for passingthrough biological barriers; 2) reproducible biodegradability withoutthe addition of enzymes or cofactors; 3) capability for sustainedrelease of an encapsulated, protected antigen over a period in the rangeof days to months by varying factors such as the monomer ratios orpolymer size, for example, poly(lactic acid) (PLA) to poly(glycolicacid) (PGA) copolymer ratios, potentially abrogating the boosterrequirement (Gupta, et al., Adv. Drug Deliv. Rev., 32(3):225-246 (1998);Kohn, et al., J. Immunol. Methods, 95(1):31-8 (1986); Langer, et al.,Adv. Drug Deliv. Rev., 28(1):97-119 (1997); Jiang, et al., Adv. DrugDeliv. Rev., 57(3):391-410)), well-understood fabrication methodologiesthat offer flexibility over the range of parameters that can be used forfabrication, including choices of the polymer material, solvent,stabilizer, and scale of production; and 5) control over surfaceproperties facilitating the introduction of modular functionalities intothe surface.

Examples of preferred biodegradable polymers include synthetic polymersthat degrade by hydrolysis such as poly(hydroxy acids), such as polymersand copolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyesters, polyurethanes, poly(butic acid),poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), andpoly(lactide-co-caprolactone).

Preferred natural polymers include alginate and other polysaccharides,collagen, albumin and other hydrophilic proteins, zein and otherprolamines and hydrophobic proteins, copolymers and mixtures thereof. Ingeneral, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion.

In some embodiments, non-biodegradable polymers can be used, especiallyhydrophobic polymers. Examples of preferred non-biodegradable polymersinclude ethylene vinyl acetate, poly(meth) acrylic acid, copolymers ofmaleic anhydride with other unsaturated polymerizable monomers,poly(butadiene maleic anhydride), polyamides, copolymers and mixturesthereof, and dextran, cellulose and derivatives thereof.

Other suitable biodegradable and non-biodegradable polymers include, butare not limited to, polyanhydrides, polyamides, polycarbonates,polyalkylenes, polyalkylene oxides such as polyethylene glycol,polyalkylene terepthalates such as poly(ethylene terephthalate),polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyethylene,polypropylene, poly(vinyl acetate), poly vinyl chloride, polystyrene,polyvinyl halides, polyvinylpyrrolidone, polymers of acrylic andmethacrylic esters, polysiloxanes, polyurethanes and copolymers thereof,modified celluloses, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, cellulose acetate, cellulosepropionate, cellulose acetate butyrate, cellulose acetate phthalate,carboxyethyl cellulose, cellulose triacetate, cellulose sulfate sodiumsalt, and polyacrylates such as poly(methyl methacrylate),poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexylmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate).

The foregoing materials may be used alone, as physical mixtures(blends), or as co-polymers. In a preferred embodiment, the nanoparticleis formed of polymers fabricated from polylactides (PLA) and copolymersof lactide and glycolide (PLGA). These have established commercial usein humans and have a long safety record (Jiang, et al., Adv. Drug Deliv.Rev., 57(3):391-410); Aguado and Lambert, Immunobiology, 184(2-3):113-25(1992); Bramwell, et al., Adv. Drug Deliv. Rev., 57(9):1247-65 (2005)).

The polymer may be a bioadhesive polymer that is hydrophilic orhydrophobic. Hydrophilic polymers include CARBOPOL™ (a high molecularweight, crosslinked, acrylic acid-based polymers manufactured byNOVEON™), polycarbophil, cellulose esters, and dextran.

Rate controlling polymers may be included in the polymer matrix or inthe coating on the formulation. Examples of rate controlling polymersthat may be used are hydroxypropylmethylcellulose (HPMC) withviscosities of either 5, 50, 100 or 4000 cps or blends of the differentviscosities, ethylcellulose, methylmethacrylates, such as EUDRAGIT®RS100, EUDRAGIT® RL100, EUDRAGIT® NE 30D (supplied by Rohm America).Gastrosoluble polymers, such as EUDRAGIT® E100 or enteric polymers suchas EUDRAGIT® L100-55D, L100 and 5100 may be blended with ratecontrolling polymers to achieve pH dependent release kinetics. Otherhydrophilic polymers such as alginate, polyethylene oxide,carboxymethylcellulose, and hydroxyethylcellulose may be used as ratecontrolling polymers.

These polymers can be obtained from sources such as Sigma Chemical Co.,St. Louis, Mo.; Polysciences, Warrenton, Pa.; Aldrich, Milwaukee, Wis.;Fluka, Ronkonkoma, N.Y.; and BioRad, Richmond, Calif., or can besynthesized from monomers obtained from these or other suppliers usingstandard techniques.

B. Antigens

Antigens can be peptides, proteins, polysaccharides, saccharides,lipids, glycolipids, nucleic acids, or combinations thereof. The antigencan be derived from ant source, including, but not limited to, a virus,bacterium, parasite, plant, protozoan, fungus, tissue or transformedcell such as a cancer or leukemic cell and can be a whole cell orimmunogenic component thereof, e.g., cell wall components or molecularcomponents thereof.

Suitable antigens are known in the art and are available from commercialgovernment and scientific sources. In one embodiment, the antigens arewhole inactivated or attenuated organisms. These organisms may beinfectious organisms, such as viruses, parasites and bacteria. Theseorganisms may also be tumor cells. The antigens may be purified orpartially purified polypeptides derived from tumors or viral orbacterial sources. Criteria for identifying and selecting effectiveantigenic peptides (e.g., minimal peptide sequences capable of elicitingan immune response) can be found in the art. For example,Apostolopoulos, et al. (Curr. Opin. Mol. Ther., 2:29-36 (2000)),discusses the strategy for identifying minimal antigenic peptidesequences based on an understanding of the three-dimensional structureof an antigen-presenting molecule and its interaction with both anantigenic peptide and T-cell receptor. Shastri, (Curr. Opin. Immunol.,8:271-7 (1996)), disclose how to distinguish rare peptides that serve toactivate T cells from the thousands peptides normally bound to MHCmolecules. The antigens can be recombinant polypeptides produced byexpressing DNA encoding the polypeptide antigen in a heterologousexpression system. The antigens can be DNA encoding all or part of anantigenic protein. The DNA may be in the form of vector DNA such asplasmid DNA.

Antigens may be provided as single antigens or may be provided incombination. Antigens may also be provided as complex mixtures ofpolypeptides or nucleic acids.

i. Viral Antigens

A viral antigen can be isolated from any virus including, but notlimited to, a virus from any of the following viral families:Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus,Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae,Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus,Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acuterespiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae,Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virusand Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)),Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2,Dengue virus 3, Dengue virus 4, and West Nile virus), Hepadnaviridae,Herpesviridae (e.g., Human herpesvirus 1, 3, 4 (Epstein-Barr virus), 5,and 6, and Cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae,Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., Influenzavirus Aand B and C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, andhuman respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g.,poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g.,vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae(e.g., lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV2), Rhabdoviridae (for example, rabies virus, measles virus, respiratorysyncytial virus, etc.), Togaviridae (for example, rubella virus, denguevirus, etc.), and Totiviridae. Suitable viral antigens also include allor part of Dengue protein M, Dengue protein E, Dengue D1NS1, DengueD1NS2, and Dengue D1NS3.

Viral antigens may be derived from a particular strain such as apapilloma virus, a herpes virus, i.e. herpes simplex 1 and 2; ahepatitis virus, for example, hepatitis A virus (HAV), hepatitis B virus(HBV), hepatitis C virus (HCV), the delta hepatitis D virus (HDV),hepatitis E virus (HEV) and hepatitis G virus (HGV), the tick-borneencephalitis viruses; parainfluenza, varicella-zoster, cytomeglavirus,Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackieviruses,equine encephalitis, Japanese encephalitis, yellow fever, Rift Valleyfever, and lymphocytic choriomeningitis.

ii. Bacterial Antigens

Bacterial antigens can originate from any bacteria including, but notlimited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio,Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium,Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus,Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus,Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella,Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium,Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria,Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas,Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum,Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus,Thermoplasma, Thiobacillus, and Treponema, Vibrio, and Yersinia.

iii. Parasite Antigens

Parasite antigens can be obtained from parasites such as, but notlimited to, an antigen derived from Cryptococcus neoformans, Histoplasmacapsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides,Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae,Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum,Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii,Trichomonas vaginalis and Schistosoma mansoni. These include Sporozoanantigens, Plasmodian antigens, such as all or part of a Circumsporozoiteprotein, a Sporozoite surface protein, a liver stage antigen, an apicalmembrane associated protein, or a Merozoite surface protein.

iv. Allergens and Environmental Antigens

The antigen can be an allergen or environmental antigen, such as, butnot limited to, an antigen derived from naturally occurring allergenssuch as pollen allergens (tree-, herb, weed-, and grass pollenallergens), insect allergens (inhalant, saliva and venom allergens),animal hair and dandruff allergens, and food allergens. Important pollenallergens from trees, grasses and herbs originate from the taxonomicorders of Fagales, Oleales, Pinales and platanaceae including, i.e.,birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) andolive (Olea), cedar (Cryptomeriaand Juniperus), Plane tree (Platanus),the order of Poales including, i.e., grasses of the genera Lolium,Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum,the orders of Asterales and Urticales including, i.e., herbs of thegenera Ambrosia, Artemisia, and Parietaria. Other allergen antigens thatmay be used include allergens from house dust mites of the genusDermatophagoides and Euroglyphus, storage mite, e.g., Lepidoglyphys,Glycyphagus and Tyrophagus, those from cockroaches, midges and flease.g., Blatella, Periplaneta, Chironomus and Ctenocepphalides, those frommammals such as cat, dog and horse, birds, venom allergens includingsuch originating from stinging or biting insects such as those from thetaxonomic order of Hymenoptera including bees (superfamily Apidae),wasps (superfamily Vespidea), and ants (superfamily Formicoidae). Stillother allergen antigens that may be used include inhalation allergensfrom fungi such as from the genera Alternaria and Cladosporium.

v. Tumor Antigens

The antigen can be a tumor antigen, including a tumor-associated ortumor-specific antigen, such as, but not limited to SOX2,alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27,cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusionprotein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-All,hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I,OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, Triosephosphateisomeras, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1,Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, andTRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2,MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE),SCP-1, Hom/Me1-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL,H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, humanpapillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA,PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG,BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50,CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344,MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP,and TPS.

C. Adaptor Elements

Adaptor elements associate with the nanoparticle and provide substratesthat facilitate the modular assembly and disassembly of functionalelements to the nanoparticle. Adaptor elements may associate withnanoparticles through a variety of interactions including, but notlimited to, hydrophobic interactions, electrostatic interactions andcovalent coupling.

In a preferred embodiment, the adaptor elements associate with thepolymeric nanoparticles noncovalently through hydrophobic interactions.Examples of adaptor elements which may associate with nanoparticles viahydrophobic interactions include, but are not limited to, fatty acids,hydrophobic or amphipathic peptides or proteins, and polymers. Theseclasses of adaptor elements may also be used in any combination orratio. In a preferred embodiment, the association of adaptor elementswith nanoparticles facilitates a prolonged presentation of functionalelements which can last for several weeks.

Adaptor elements can also be attached to polymeric nanoparticles throughcovalent interactions through various functional groups. Functionalityrefers to conjugation of a molecule to the surface of the particle via afunctional chemical group (carboxylic acids, aldehydes, amines,sulfhydryls and hydroxyls) present on the surface of the particle andpresent on the molecule to be attached.

Functionality may be introduced into the particles in two ways. Thefirst is during the preparation of the nanoparticles, for example duringthe emulsion preparation of nanoparticles by incorporation ofstabilizers with functional chemical groups. Suitable stabilizersinclude hydrophobic or amphipathic molecules that associate with theouter surface of the nanoparticles.

A second is post-particle preparation, by direct crosslinking particlesand ligands with homo- or heterobifunctional crosslinkers. This secondprocedure may use a suitable chemistry and a class of crosslinkers (CDI,EDAC, glutaraldehydes, etc. as discussed in more detail below) or anyother crosslinker that couples ligands to the particle surface viachemical modification of the particle surface after preparation. Thissecond class also includes a process whereby amphiphilic molecules suchas fatty acids, lipids or functional stabilizers may be passivelyadsorbed and adhered to the particle surface, thereby introducingfunctional end groups for tethering to ligands.

One useful protocol involves the “activation” of hydroxyl groups onpolymer chains with the agent, carbonyldiimidazole (CDI) in aproticsolvents such as DMSO, acetone, or THF. CDI forms an imidazolylcarbamate complex with the hydroxyl group which may be displaced bybinding the free amino group of a molecule such as a protein. Thereaction is an N-nucleophilic substitution and results in a stableN-alkylcarbamate linkage of the molecule to the polymer. The “coupling”of the molecule to the “activated” polymer matrix is maximal in the pHrange of 9-10 and normally requires at least 24 hrs. The resultingmolecule-polymer complex is stable and resists hydrolysis for extendedperiods of time.

Another coupling method involves the use of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or “water-solubleCDI” in conjunction with N-hydroxylsulfosuccinimide (sulfo NHS) tocouple the exposed carboxylic groups of polymers to the free aminogroups of molecules in a totally aqueous environment at thephysiological pH of 7.0. Briefly, EDAC and sulfo-NHS form an activatedester with the carboxylic acid groups of the polymer which react withthe amine end of a molecule to form a peptide bond. The resultingpeptide bond is resistant to hydrolysis. The use of sulfo-NHS in thereaction increases the efficiency of the EDAC coupling by a factor often-fold and provides for exceptionally gentle conditions that ensurethe viability of the molecule-polymer complex.

By using either of these protocols it is possible to “activate” almostall polymers containing either hydroxyl or carboxyl groups in a suitablesolvent system that will not dissolve the polymer matrix.

A useful coupling procedure for attaching molecules with free hydroxyland carboxyl groups to polymers involves the use of the cross-linkingagent, divinylsulfone. This method would be useful for attaching sugarsor other hydroxylic compounds with bioadhesive properties to hydroxylicmatrices. Briefly, the activation involves the reaction ofdivinylsulfone to the hydroxyl groups of the polymer, forming thevinylsulfonyl ethyl ether of the polymer. The vinyl groups will coupleto alcohols, phenols and even amines. Activation and coupling take placeat pH 11. The linkage is stable in the pH range from 1-8 and is suitablefor transit through the intestine.

Any suitable coupling method known to those skilled in the art for thecoupling of molecules and polymers with double bonds, including the useof UV crosslinking, may be used for attachment of molecules to thepolymer.

In one embodiment adaptor elements can be conjugated to affinity tags.Affinity tags are any molecular species which form highly specific,noncovalent, physiochemical interactions with defined binding partners.Affinity tags which form highly specific, noncovalent, physiochemicalinteractions with one another are defined herein as “complementary”.Suitable affinity tag pairs are well known in the art and includeepitope/antibody, biotin/avidin, biotin/streptavidin,biotin/neutravidin, glutathione-S-transferase/glutathione, maltosebinding protein/amylase and maltose binding protein/maltose. Examples ofsuitable epitopes which may be used for epitope/antibody binding pairsinclude, but are not limited to, HA, FLAG, c-Myc,glutatione-S-transferase, His₆, GFP, DIG, biotin and avidin. Antibodies(both monoclonal and polyclonal and antigen-binding fragments thereof)which bind to these epitopes are well known in the art.

Affinity tags that are conjugated to adaptor elements allow for highlyflexible, modular assembly and disassembly of functional elements whichare conjugated to affinity tags which form highly specific, noncovalent,physiochemical interactions with complementary affinity tags which areconjugated to adaptor elements. Adaptor elements may be conjugated witha single species of affinity tag or with any combination of affinity tagspecies in any ratio. The ability to vary the number of species ofaffinity tags and their ratios conjugated to adaptor elements allows forexquisite control over the number of functional elements which may beattached to the nanoparticles and their ratios.

In another embodiment adaptor elements are coupled directly tofunctional elements in the absence of affinity tags, such as throughdirect covalent interactions. Adaptor elements can be covalently coupledto at least one species of functional element. Adaptor elements can becovalently coupled to a single species of functional element or with anycombination of species of functional elements in any ratio.

In a preferred embodiment adaptor elements are conjugated to at leastone affinity tag that provides for assembly and disassembly of modularfunctional elements which are conjugated to complementary affinity tags.In a more preferred embodiment, adaptor elements are fatty acids thatare conjugated with at least one affinity tag. In a particularlypreferred embodiment, the adaptor elements are fatty acids conjugatedwith avidin or streptavidin. Such avidin/streptavidin-conjugated fattyacids allow for the attachment of a wide variety of biotin-conjugatedfunctional elements.

The adaptor elements are provided on, or in the surface of,nanoparticles at a high density. This high density of adaptor elementsallows for coupling of the nanoparticle to a variety of species offunctional elements while still allowing for the functional elements tobe present in high enough numbers to be efficacious.

i. Fatty Acids

The adaptor elements may include fatty acids. Fatty acids may be of anyacyl chain length and may be saturated or unsaturated. In a particularlypreferred embodiment the fatty acid is palmitic acid. Other suitablefatty acids include, but are not limited to, saturated fatty acids suchas butyric, caproic, caprylic, capric, lauric, myristic, stearic,arachidic and behenic acid. Still other suitable fatty acids include,but are not limited to, unsaturated fatty acids such as oleic, linoleic,alpha-linolenic, arachidonic, eicosapentaenoic, docosahexaenoic anderucic acid.

ii. Hydrophobic or Amphipathic Peptides

The adaptor elements may include hydrophobic or amphipathic peptides.Preferred peptides should be sufficiently hydrophobic to preferentiallyassociate with the polymeric nanoparticle over the aqueous environment.Amphipathic polypeptides useful as adaptor elements may be mostlyhydrophobic on one end and mostly hydrophilic on the other end. Suchamphipathic peptides may associate with polymeric nanoparticles throughthe hydrophobic end of the peptide and be conjugated on the hydrophilicend to a functional group.

iii. Hydrophobic Polymers

Adaptor elements may include hydrophobic polymers. Examples ofhydrophobic polymers include, but are not limited to, polyanhydrides,poly(ortho)esters, and polyesters such as polycaprolactone.

D. Functional Elements

Functional elements which associate with the nanoparticles provide anumber of different functions to the composition. These functionsinclude protection of the nanoparticle vaccine from hostile environmentsduring transit in the gastrointestinal tract, transport throughepithelial barriers, targeting antigen presenting cells (APCs) with highavidity, and transport of mediators that facilitate uptake andpresentation of antigen by antigen-presenting cells through disruptionof intracellular antigen-sequestering compartments. Functional elementsmay include dendritic cell recognition elements, epithelial cellrecognition elements, pH-sensitive molecules which protect thecomposition from hydrolysis and degradation in low-pH environments,non-pH-sensitive molecules which protect the composition from hydrolysisand degradation in low-pH environments, and/or endosome-disruptingagents.

Nanoparticles may be associated with a single species of functionalelement or may be associated with any combination of disclosedfunctional elements in any ratio. In one embodiment, functional elementsare directly associated with nanoparticles in the absence of adaptorelements. Functional elements may be directly associated withnanoparticles through covalent or noncovalent interactions, including,but not limited to, hydrophobic interactions and electrostaticinteractions. Covalent attachment of functional elements can be achievedby introducing functionality to the polymeric nanoparticles usingmethods described above with respect to adaptor elements.

In another embodiment, functional elements are associated withnanoparticles through adaptor elements which directly associate with thenanoparticles. Functional elements may be directly, covalently coupledto adaptor elements or may couple to adaptor elements throughcomplementary affinity tags conjugated to the adaptor and functionalelements. Multiple different species of functional elements may beassociated with nanoparticles in any desired ratio, for instance, byconjugating each species of functional element to a separate species ofaffinity tag. These functional elements may then associate withnanoparticles coated with adaptor elements conjugated to an appropriateratio of complementary affinity tags. Multiple species of functionalelements may also be associated with nanoparticles by covalentlycoupling each species of functional element at a desired ratio toadaptor elements.

In a preferred embodiment, functional elements are conjugated to biotin.Biotin conjugation allows the functional elements to interact withadaptor elements conjugated with avidin, neutravidin or streptavidin.

i. Targeting Molecules for Professional Antigen Presenting Cells

Of the main types of antigen-presenting cells (B cell, macrophages andDCs), the DC is the most potent and is responsible for initiating allantigen-specific immune responses. One biological feature of DCs istheir ability to sense conditions under which antigen is encountered,initiating a process of “DC maturation”. Using receptors for variousmicrobial and inflammatory products, DCs respond to antigen exposure indifferent ways depending on the nature of the pathogen (virus, bacteria,protozoan) encountered. This information is transmitted to T cells byaltered patterns of cytokine release at the time of antigen presentationin lymph nodes, altering the type of T cell response elicited. Thus,targeting DCs provides the opportunity not only to quantitativelyenhance the delivery of antigen and antigen responses in general, but toqualitatively control the nature of the immune response depending on thedesired vaccination outcome.

Dendritic cells express a number of cell surface receptors that canmediate the endocytosis of bound antigen. Targeting exogenous antigensto internalizing surface molecules on systemically-distributed antigenpresenting cells facilitates uptake of antigens and thus overcomes amajor rate-limiting step in immunization and thus in vaccination.

Dendritic cell targeting molecules include monoclonal or polyclonalantibodies or fragments thereof that recognize and bind to epitopesdisplayed on the surface of dendritic cells. Dendritic cell targetingmolecules also include ligands which bind to a cell surface receptor ondendritic cells. One such receptor, the lectin DEC-205, has been used invitro and in mice to boost both humoral (antibody-based) and cellular(CD8 T cell) responses by 2-4 orders of magnitude (Hawiger, et al., J.Exp. Med., 194(6):769-79 (2001); Bonifaz, et al., J. Exp. Med.,196(12):1627-38 (2002); Bonifaz, et al., J. Exp. Med., 199(6):815-24(2004)). In these experiments, antigens were fused to an anti-DEC205heavy chain and a recombinant antibody molecule was used forimmunization.

A variety of other endocytic receptors, including a mannose-specificlectin (mannose receptor) and IgG Fc receptors, have also been targetedin this way with similar enhancement of antigen presentation efficiency.Other suitable molecules which may be targeted include, but are notlimited to, DC-SIGN, BDCA3 (CD141), 33D1, SIGLEC-H, DCIR, CD11c, heatshock protein receptors and scavenger receptors.

Other receptors which may be targeted include the toll-like receptors(TLRs). TLRs recognize and bind to pathogen-associated molecularpatterns (PAMPs). PAMPs target the TLR on the surface of the dendriticcell and signals internally, thereby potentially increasing DC antigenuptake, maturation and T-cell stimulatory capacity. PAMPs conjugated tothe particle surface or co-encapsulated include unmethylated CpG DNA(bacterial), double-stranded RNA (viral), lipopolysacharride(bacterial), peptidoglycan (bacterial), lipoarabinomannin (bacterial),zymosan (yeast), mycoplasmal lipoproteins such as MALP-2 (bacterial),flagellin (bacterial) poly(inosinic-cytidylic) acid (bacterial),lipoteichoic acid (bacterial) or imidazoquinolines (synthetic).

ii. Targeting Molecules for Epithelial Cells

The potential efficacy of nanoparticle vaccine systems is determined inpart by their route of administration into the body. While injection(intradermal, intramuscular, intravenous) is an acceptable solution inmany cases, having a vaccine product that is orally available willgreatly extend its ease of use and applicability on a global scale. Fororally administered vaccines, epithelial cells constitute the principalbarrier that separates an organism's interior from the outside world.Epithelial cells such as those that line the gastrointestinal tract formcontinuous monolayers that simultaneously confront the extracellularfluid compartment and the extracorporeal space. Uptake by these gutepithelial cells can be enhanced, and the nanoparticles carried by“transcytosis” to the lymphatics where they have access to dendriticcells.

Through the process of “antigen sampling”, underlying mucosal-associatedlymphoid tissue sample the environment for the presence of pathogens.This sampling is carried out by an apical to basolateral transcytoticevent and is mediated by M cells located in lymphoid follicle-associatedepithelium (FAE) throughout the GI tract. In addition, absorptiveenterocytes may transport microorganisms or other nanoparticulates tointraepithelial lymphocytes. DCs may perform this function directly,with a population of DCs being intercalated between epithelial cells andextending processes into the gut lumen to sample the microorganismspresent.

Adherence to cells is an essential first step in crossing the epithelialbarrier by any of these mechanisms. Therefore, in one embodiment,modular nanoparticle vaccines further include epithelial cellrecognition elements. Epithelial cell targeting molecules includemonoclonal or polyclonal antibodies or bioactive fragments thereof thatrecognize and bind to epitopes displayed on the surface of epithelialcells. Epithelial cell targeting molecules also include ligands whichbind to a cell surface receptor on epithelial cells. Ligands include,but are not limited to, molecules such as polypeptides, nucleotides andpolysaccharides.

A variety of receptors on epithelial cells may be targeted by epithelialcell targeting molecules. Examples of suitable receptors to be targetedinclude, but are not limited to, IgE Fc receptors, EpCAM, selectedcarbohydrate specificities, dipeptidyl peptidase, and E-cadherin.

iii. Coatings to Inhibit Degradation of Nanoparticle VaccineCompositions in Extreme pH Environments

Vaccine particles administered orally will encounter a corrosiveenvironment in the gastrointestinal (GI) tract with areas of low andhigh pH, as well as resident degradative enzymes and solubilizingagents. Biodegradable particulates have gained attention as oralvaccines because of their ability to protect antigens on route to immunesites across the intestinal epithelium (O'Hagan and Valiante, Nat. Rev.Drug Discov., 2(9):727-35 (2003); van der Lubben, et al., Adv. DrugDeliv. Rev., 52(2):139-44 (2001); Wikingsson and Sjoholm, Vaccine,20(27-28):3355-63 (2002); Moser, et al., Expert Rev. Vaccines,2(2):189-96 (2003)). However, while the antigen is protected fromenvironmental elements in transit, little protection is offered toelements coupled to the surface of the particle during the transit toimmune effector sites. This protection may be necessary to insure properparticle function and targeting.

For this reason, ‘shielding’ is a desired feature to protect thenanoparticulate and its immune recognition elements in transit to the GIepithelium. This shielding may be environmentally-sensitive, i.e. pHresponsive, or simply a protective layer. In a preferred embodiment,modular nanoparticle vaccines further include pH-sensitive moleculeswhich protect the composition from hydrolysis and degradation in low pHenvironments. Such pH-sensitive protecting molecules are preferredbecause subsequent to the particles transit through a low pHenvironment, upon reaching its destination in the higher pH intestinalsite, particles should expose epithelial targeting molecules to allowfor specific interactions with target epithelial cells, followed bytranscytosis through the epithelium and subsequent interactions withsubepithelial dendritic cells.

Preferred non-pH-sensitive molecules which protect the composition fromhydrolysis and degradation in low pH environments are poly(ethylene)glycol, gelatin and albumins.

Preferred pH-sensitive molecules which protect the composition fromhydrolysis and degradation in low pH environments are elastin andpoly(methacrylic) acid (PMAA). Both of these molecules are in extendedconformations at pH 7.4 and shrink rapidly (within seconds) uponexposure to lower pH environments (below pH 5 for elastin and below pH5-6 for poly(methacylic) acid). Other pH-sensitive molecules which maybe used include poly(acrylic acid), poly(methyl methacrylic acid) andpoly(N-alkyl acrylamides), or other enteric coatings discussed above.

pH-sensitive or pH-insensitive protective molecules may be directlycoupled to nanoparticles, or may be coupled to nanoparticles throughadaptor elements, such as those described above. In a preferredembodiment the epithelial cell targeting molecules are functionallycoupled to adaptor elements.

E. Disruptors of Endosomal Compartments

Many receptors which may be used for targeting modular nanoparticlevaccines to dendritic cells, such as DEC-205, have the property ofdelivering antigens to late endosomal elements that serve as efficientsites for the formation of immunogenic peptides and their loading ontoMHC class II molecules (which are needed for CD4 T cell and antibodyresponses) (Mellman, Adv. Exp. Med. Biol., 560:63-7; Mellman andSteinman, Cell, 106(3):255-8 (2001)). Effective vaccination, however,often requires the production of CD8 cytotoxic T cell responses whichoccurs only when antigen is present in the cytoplasm. DCs are adept atthis function by the process of “cross-presentation”, whereby exogenousantigens escape endocytic vesicles and enter the cytoplasm where theyare cleaved into peptides by the proteasome, imported into theendoplasmic reticulum, and loaded onto newly synthesized MHC class Imolecules (which are required for stimulation of CD8 T cells).

It is possible to greatly enhance the efficiency of cross presentationby artificially causing the limited disruption of endosome-lysosomemembranes during antigen uptake. In one embodiment, the modularnanoparticulate vaccine compositions include an agent which causes thedisruption of endosomal membranes. Endosomal membrane disrupting agentsinclude, but are not limited to, small molecule drugs, peptides,polypeptides, including elastin, and synthetic agents that disruptintracellular pH or vesicular membranes.

Osmotic delivery is an endocytosis-mediated system for delivering largepolar molecules into the cytosol of cells. The mechanism behind thisprocess is also referred to as the “proton-sponge effect”. The processrenders the endosome fragile, releasing the contents of the endosomeinto the cytosol. Osmotic delivery and endosomal disruption work asfollows: uptake of high-osmolarity cargo (typically charged substances)into newly forming endosomes renders their membranes fragile, which uponreturn to normal osmolarity after uptake, causes a net inflow of waterinto loaded endosomes resulting in build-up of pressure. This leads toan osmotic pressure within the endosomes and release of the cargo intothe cytosol of the cell.

In a preferred embodiment, the endosome-disrupting agent is a lowpH-activated, amphipathic, pore-forming peptide. In a particularlypreferred embodiment, the low pH-activated, amphipathic, pore-formingpeptide is the commercially-available Endoporter (Endoporter; GeneTools,Philomath, OR) (Summerton, Ann. N.Y. Acad. Sci., 1058:1-14 (2005)).

Agents other than Endoporter can mediate endosome disruption. Poly(lactic-co-glycolic acid) (PLGA) on its own can mediate this effect.This process can be increased by inclusion of other agents in PLGA.Other agents include charged macromolecules such as poly(amido amine)(PAMAM) dendrimers, or small molecule drugs such as carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP), and oligonucleotides such asCpG. The endosome-disrupting agent may be encapsulated into thepolymeric core of the nanoparticle. Additionally, or alternatively, theendosome-disrupting agent may be attached to the surface of thenanoparticle by association with attached adaptor elements.

G. Adjuvants

The modular nanoparticulate vaccines can include adjuvants. These can beincorporated into, administered with, or administered separately from,the vaccine nanoparticles. Adjuvants may be provided encapsulated orotherwise entrapped in the polymeric core of the nanoparticle vaccine,or may be associated with the surface of the nanoparticle either throughdirect association with the polymeric core, or through association withadaptor elements. Adjuvant may be in the form of separate nanoparticlesor in a suspension or solution administered with the vaccinenanoparticles.

In one embodiment the adjuvant is the synthetic glycolipidalpha-galactosylceramide (αGalCer). Dendritic cells presenting antigensin the context of CD1d can lead to rapid innate and prolonged productionof cytokines such as interferon and IL-4 by natural killer T cells (NKTcells). CD1d is a major histocompatibility complex class I-like moleculethat presents glycolipid antigens to a subset of NKT cells.Advantageously, αGalCer is not toxic to humans and has been shown to actas an adjuvant, priming both antigen-specific CD4+ and CD8+ T cellresponses. For example, it has been shown that αGalCer in conjunctionwith a malaria vaccine can lead to cytotoxic responses against infectedcells, which is an ideal scenario for vaccines against infectiousdiseases. In addition to αGalCer, other glycolipids that function asadjuvants to activate NKT cell-mediated immune responses can be used.

In another embodiment the adjuvant can be, but is not limited to, one ormore of the following: oil emulsions (e.g., Freund's adjuvant); saponinformulations; virosomes and viral-like particles; bacterial andmicrobial derivatives including, but not limited to, carbohydrates suchas lipopolysachharide (LPS); immunostimulatory oligonucleotides;ADP-ribosylating toxins and detoxified derivatives; alum; BCG;mineral-containing compositions (e.g., mineral salts, such as aluminiumsalts and calcium salts, hydroxides, phosphates, sulfates, etc.);bioadhesives and/or mucoadhesives; microparticles; liposomes;polyoxyethylene ether and polyoxyethylene ester formulations;polyphosphazene; muramyl peptides; imidazoquinolone compounds; andsurface active substances (e.g. lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol).

Adjuvants may also include immunomodulators such as cytokines,interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.),interferons (e.g., interferon-.gamma.), macrophage colony stimulatingfactor, and tumor necrosis factor; and co-stimulatory molecules, such asthose of the B7 family. Such proteinaceous adjuvants may be provided asthe full-length polypeptide or an active fragment thereof, or in theform of DNA, such as plasmid DNA.

H. Contrast Agents and Other Markers

Optionally, modular nanoparticulate vaccine may further include agentsuseful for determining the location of administered particles. Agentsuseful for this purpose include fluorescent tags, radionuclides andcontrast agents.

Suitable imaging agents include, but are not limited to, fluorescentmolecules such as those described by Molecular Probes (Handbook offluorescent probes and research products), such as Rhodamine,fluorescein, Texas red, Acridine Orange, Alexa Fluor (various),Allophycocyanin, 7-aminoactinomycin D, BOBO-1, BODIPY (various),Calcien, Calcium Crimson, Calcium green, Calcium Orange,6-carboxyrhodamine 6G, Cascade blue, Cascade yellow, DAPI, DiA, DiD,Dil, DiO, DiR, ELF 97, Eosin, ER Tracker Blue-White, EthD-1, Ethidiumbromide, Fluo-3, Fluo4, FM1-43, FM4-64, Fura-2, Fura Red, Hoechst 33258,Hoechst 33342, 7-hydroxy-4-methylcoumarin, Indo-1, JC-1, JC-9, JOE dye,Lissamine rhodamine B, Lucifer Yellow CH, LysoSensor Blue DND-167,LysoSensor Green, LysoSensor Yellow/Blu, Lysotracker Green FM, MagnesiumGreen, Marina Blue, Mitotracker Green FM, Mitotracker Orange CMTMRos,MitoTracker Red CMXRos, Monobromobimane, NBD amines, NeruoTrace 500/525green, Nile red, Oregon Green, Pacific Blue. POP-1, Propidium iodide,Rhodamine 110, Rhodamine Red, R-Phycoerythrin, Resorfin, RH414, Rhod-2,Rhodamine Green, Rhodamine 123, ROX dye, Sodium Green, SYTO blue(various), SYTO green (Various), SYTO orange (various), SYTOX blue,SYTOX green, SYTOX orange, Tetramethylrhodamine B, TOT-1, TOT-3,X-rhod-1, YOYO-1, YOYO-3.

Additionally radionuclides can be used as imaging agents. Suitableradionuclides include, but are not limited to radioactive species ofFe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(I1) Indium, Gallium andTechnetium. Other suitable contrast agents include metal ions generallyused for chelation in paramagnetic T1-type MIR contrast agents, andinclude di- and tri-valent cations such as copper, chromium, iron,gadolinium, manganese, erbium, europium, dysprosium and holmium. Metalions that can be chelated and used for radionuclide imaging, include,but are not limited to metals such as gallium, germanium, cobalt,calcium, indium, iridium, rubidium, yttrium, ruthenium, yttrium,technetium, rhenium, platinum, thallium and samarium. Additionally metalions known to be useful in neutron-capture radiation therapy includeboron and other metals with large nuclear cross-sections. Also suitableare metal ions useful in ultrasound contrast, and X-ray contrastcompositions.

Examples of other suitable contrast agents include gases or gas emittingcompounds, which are radioopaque.

I. Pharmaceutically Acceptable Excipients

The compositions may be administered in combination with aphysiologically or pharmaceutically acceptable carrier, excipient, orstabilizer. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients. The term“pharmaceutically-acceptable carrier” means one or more compatible solidor liquid fillers, dilutants or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm “carrier” refers to an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application.

Pharmaceutical compositions may be formulated in a conventional mannerusing one or more physiologically acceptable carriers includingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.

Optional pharmaceutically acceptable excipients include, but are notlimited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants. Diluents, also referred to as “fillers,”are typically necessary to increase the bulk of a solid dosage form sothat a practical size is provided for compression of tablets orformation of beads and granules. Suitable diluents include, but are notlimited to, dicalcium phosphate dihydrate, calcium sulfate, lactose,sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose,kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinizedstarch, silicone dioxide, titanium oxide, magnesium aluminum silicateand powdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (POLYPLASDONE® XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard decomposition reactions whichinclude, by way of example, oxidative reactions.

Surfactants may be anionic, cationic, amphoteric or nonionic surfaceactive agents. Suitable anionic surfactants include, but are not limitedto, those containing carboxylate, sulfonate and sulfate ions. Examplesof anionic surfactants include sodium, potassium, ammonium of long chainalkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-b-alanine, sodium N-lauryl-b-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

If desired, the particles may also contain minor amount of nontoxicauxiliary substances such as wetting or emulsifying agents, dyes, pHbuffering agents, or preservatives.

The particles may be complexed with other agents. The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., acacia, methylcellulose, sodiumcarboxymethylcellulose, polyvinylpyrrolidone (Povidone), hydroxypropylmethylcellulose, sucrose, starch, and ethylcellulose); fillers (e.g.,corn starch, gelatin, lactose, acacia, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, calcium carbonate,sodium chloride, or alginic acid); lubricants (e.g. magnesium stearates,stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica);and disintegrators (e.g. micro-crystalline cellulose, corn starch,sodium starch glycolate and alginic acid. If water-soluble, suchformulated complex then may be formulated in an appropriate buffer, forexample, phosphate buffered saline or other physiologically compatiblesolutions. Alternatively, if the resulting complex has poor solubilityin aqueous solvents, then it may be formulated with a non-ionicsurfactant such as TWEEN™, or polyethylene glycol. Thus, the compoundsand their physiologically acceptable solvates may be formulated foradministration.

Liquid formulations for oral administration prepared in water or otheraqueous vehicles may contain various suspending agents such asmethylcellulose, alginates, tragacanth, pectin, kelgin, carrageenan,acacia, polyvinylpyrrolidone, and polyvinyl alcohol. The liquidformulations may also include solutions, emulsions, syrups and elixirscontaining, together with the active compound(s), wetting agents,sweeteners, and coloring and flavoring agents. Various liquid and powderformulations can be prepared by conventional methods for inhalation bythe patient.

The particles may be further coated. Suitable coating materials include,but are not limited to, cellulose polymers such as cellulose acetatephthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose,hydroxypropyl methylcellulose phthalate and hydroxypropylmethylcellulose acetate succinate; polyvinyl acetate phthalate, acrylicacid polymers and copolymers, and methacrylic resins that arecommercially available under the trade name EUDRAGIT® (Röhm Pharma,Darrnstadt, Germany), zein, shellac, and polysaccharides. Additionally,the coating material may contain conventional carriers such asplasticizers, pigments, colorants, glidants, stabilization agents, poreformers and surfactants.

III. METHODS AND MATERIALS FOR MANUFACTURE AND FORMULATION OFNANOPARTICULATE VACCINE COMPOSITIONS

A. Methods of Making Antigen-Encapsulated Nanoparticles

Many different processes can be used to form the nanoparticles. If theprocess does not produce particles having a homogenous size range, thenthe particles can be separated using standard techniques such as sievingto produce a population of particles having the desired size range.

i. Solvent Evaporation

Methods for forming nanoparticles using solvent evaporation techniquesare described in E. Mathiowitz, et al., J. Scanning Microscopy, 4:329(1990); Beck, et al., Fertil. Steril., 31:545 (1979); Beck, et al., Am.J. Obstet. Gynecol., 135(3) (1979); Benita, et al., J. Pharm. Sci.,73:1721 (1984); and U.S. Pat. No. 3,960,757 to Morishita, et al. Thepolymer is dissolved in a volatile organic solvent, such as methylenechloride. A substance to be incorporated optionally is added to thesolution, and the mixture is suspended in an aqueous solution thatcontains a surface active agent such as poly(vinyl alcohol). Substanceswhich can be incorporated in the nanoparticles include, but are notlimited to, antigens, adjuvants, imaging agents, endosome-disruptingagents and contrast agents. The resulting emulsion is stirred until mostof the organic solvent evaporated, leaving solid nano- andmicroparticles.

In a preferred embodiment, antigen-loaded, spherical PLGA nanoparticleswith a mean diameter of 100-200 nm and protein loadings of up to 40% areproduced by a modified version of this technique. In this method, 100 mgof PLGA is dissolved in 2 ml of methylene chloride in a short glass testtube (5.8 cm long, diameter 1.2 cm) overnight. To this solution,approximately 100-200 ul of the concentrated antigen solution is addedand vortexed rapidly. This solution is added drop wise to 4 ml of anaqueous solution of 5% poly (vinyl alcohol) while vortexing. Theemulsion formed is further sonicated three times for intervals of 10seconds each at 38% amplitude (Tekmar Soni Disrupter model TM300, 40%duty cycle, microtip #4) to yield a homogeneous milky mixture. Thesingle emulsion is poured into 100 ml of PVA 0.3%. The polymer/PVAdispersion is stirred on a magnetic stir plate for 3 hours at roomtemperature to allow for adequate solvent evaporation. Once solidified,the nanospheres are isolated by centrifugation (12000 rpm, 4° C., 10minutes). The supernatant is discarded. Nanospheres are washed threetimes with deionized water (10 ml) to remove excess of PVA before theyare frozen at −80° C. and then lyophilized for 48 hours. All parametersof this method are easily scaled to produce different batch sizes ofnanoparticles.

This method is useful for relatively stable polymers like polyesters andpolystyrene. However, labile polymers, such as polyanhydrides, maydegrade during the fabrication process due to the presence of water. Forthese polymers, some of the following methods performed in completelyanhydrous organic solvents are more useful.

ii. Hot Melt Microencapsulation

Microspheres can be formed from polymers such as polyesters andpolyanhydrides using hot melt microencapsulation methods as described inMathiowitz, et al., Reactive Polymers, 6:275 (1987). In this method, theuse of polymers with molecular weights between 3-75,000 daltons ispreferred. In this method, the polymer first is melted and then mixedwith the solid particles of a substance to be incorporated that havebeen sieved to less than 50 microns. The mixture is suspended in anon-miscible solvent (like silicon oil), and, with continuous stirring,heated to above the melting point of the polymer, for example, 5° C.Once the emulsion is stabilized, it is cooled until the polymerparticles solidify. The resulting microspheres are washed by decantingwith petroleum ether to give a free-flowing powder. Microspheres withsizes between one to 1000 microns are obtained with this method.

iii. Solvent Extraction

This technique is primarily designed for polyanhydrides and isdescribed, for example, in WO 93/21906 to Brown University ResearchFoundation. In this method, the substance to be incorporated isdispersed or dissolved in a solution of the selected polymer in avolatile organic solvent, such as methylene chloride. This mixture issuspended by stirring in an organic oil, such as silicon oil, to form anemulsion. Microspheres that range between 1-300 microns can be obtainedby this procedure.

iv. Spray-Drying

Methods for forming microspheres using spray drying techniques aredescribed in U.S. Pat. No. 6,620,617, to Mathiowitz et al. In thismethod, the polymer is dissolved in an organic solvent such as methylenechloride or in water. A known amount of an agent to be incorporated issuspended (insoluble agent) or co-dissolved (soluble agent) in thepolymer solution. The solution or the dispersion then is spray-dried.Microspheres ranging between 0.1-10 microns are obtained.

v. Phase Inversion

Microspheres can be formed from polymers using a phase inversion methodwherein a polymer is dissolved in a “good” solvent, fine particles of asubstance to be incorporated, such as a drug, are mixed or dissolved inthe polymer solution, and the mixture is poured into a strongnon-solvent for the polymer, to spontaneously produce, under favorableconditions, polymeric microspheres, wherein the polymer is either coatedwith the particles or the particles are dispersed in the polymer. Themethod can be used to produce microparticles in a wide range of sizes,including, for example, about 100 nanometers to about 10 microns.Exemplary polymers which can be used include polyvinylphenol andpolylactic acid. Substances which can be incorporated include, forexample, imaging agents such as fluorescent dyes, or biologically activemolecules such as proteins or nucleic acids. In the process, the polymeris dissolved in an organic solvent and then contacted with anon-solvent, which causes phase inversion of the dissolved polymer toform small spherical particles, with a narrow size distributionoptionally incorporating an antigen or other substance.

B. Methods of Attaching Adaptor Elements to Nanoparticles

Adaptor elements may be conjugated to affinity tags prior to, or aftertheir association with polymeric nanoparticles. In a preferredembodiment, the adaptor elements are fatty acids and the affinity tag isavidin/streptavidin. In a more preferred embodiment, palmitic acid isconjugated to avidin. In one method, avidin is dissolved at aconcentration of 5 mg/ml in 37° C. prewarmed 2 ml solution of 2%deoxycholate in 1×PBS. To this solution, a 10 fold molar excess ofNHS-Palmitic acid is added and the solution is stirred and sonicated in37° C. water bath (Branson, 50 kHz freq.). The reaction is maintained at37° C. for 24 hours after which excess palmitic acid is removed bydialysis against a 0.15% deoxycholate-PBS buffer prewarmed to 37° C.After three buffer changes, the avidin-palmitic acid conjugate isverified by reverse phase HPLC on a Prevail C18 column with a linearmethanol gradient in 1×PBS as the mobile phase and UV detection at 280nm. This method is easily adapted to conjugate avidin to any fatty acidof choice.

Avidin may be coupled to peptides and polymers by similar techniques.The chemistry involved in the coupling reaction will depend on thenature of available functional groups on the fatty acid, peptide orpolymer. Methods for conjugating avidin to fatty acids, peptides andpolymers are well known in the art. Methods for conjugating otheraffinity tags such as biotin, epitope tags (HA, FLAG, c-myc) andantibodies to fatty acids, peptides and polymers are well known in theart.

In a preferred embodiment, adaptor elements such as those describedabove, including fatty acids, hydrophobic or aliphatic peptides, andpolymers, are conjugated onto the surface of nanoparticles at theemulsion stage of nanoparticle preparation. In a particularly preferredembodiment, the nanoparticles include PLGA and the adaptor elementsinclude avidin-conjugated palmitic acid. In one method, dissolved PLGAsolution is added to a 4 ml solution of 2 parts avidin-palmitic acid, 2parts 5% PVA. A 50:50 mixture of protein-palmitic acid conjugates and 5%PVA has been found to yield optimal surface coverage of avidin groups onnanosized particles.

C. Methods of Attaching Functional Elements to Adaptor Elements

Functional elements can routinely be assembled onto adaptor elementsincorporated onto the nanoparticle surface by conjugating the functionalelements to affinity tags which are complementary to the affinity tagsconjugated to the adaptor elements. Especially useful affinity tag pairsfor use in coupling adaptor elements to functional elements arebiotin-avidin and biotin-streptavidin. Affinity tag-conjugatedfunctional elements are incubated with nanoparticles pre-coated withadaptor elements conjugated to complementary affinity tags under anyappropriate buffer, salt and detergent conditions. For example, typicalincubations may be performed at 4° C. for 2-4 hours, 37° C. for 20minutes or room temperature for 1 hour. Incubations may be performed inphosphate buffered saline or other buffer compositions adjusted to a pHbetween 6.0 and 7.4. Incubation may occur with gentle shaking, rockingor rotation. Nanoparticles may then be washed with excess incubationbuffer to remove unbound or non-specifically bound functional elements.

Functional elements may also be conjugated directly to adaptor elementsin the absence of affinity tags, either prior to, or after theirassociation with polymeric nanoparticles. Methods for conjugatingfunctional elements such as peptides, polypeptides, polymers andantibodies to adaptor elements such as fatty acids, peptides andpolymers are well known in the art. For example, fatty acids such aspalmitic acid may be conjugated to the C-terminus of peptides,polypeptides and antibodies using a methodology similar to thatdescribed above for conjugation of palmitic acid to avidin.

IV. METHODS OF USING NANOPARTICULATE VACCINE COMPOSITIONS

The nanoparticle vaccine compositions disclosed herein are useful foractivating T cells in subjects for prophylactic and therapeuticapplications. Activation of T cells by nanoparticle vaccine compositionsincreases their proliferation, cytokine production, differentiation,effector functions and/or survival. Methods for measuring these are wellknown to those in the art. The T cells activated by the nanoparticlevaccine compositions can be any cell which express the T cell receptor,including α/β and γ/δ T cell receptors. T-cells include all cells whichexpress CD3, including T-cell subsets which also express CD4 and CD8.Other markers of T cell subsets include KLRG1, CD127, CD44 andcombinations thereof. T-cells include both naive and memory cells andeffector cells such as CTL. T-cells also include regulatory cells suchas Th1, Tc1, Th2, Tc2, Th3, Treg, and Tr1 cells. T-cells also includeNKT-cells and similar unique classes of the T-cell lineage. In preferredembodiments the T cells that are activated are CD8⁺ T cells. Asdemonstrated in the examples below, the APCs disclosed hereinpreferentially activate and expand CD8⁺ T cells when activated ex vivo.

A. Subjects to be Treated

In general, the compositions described herein are useful for treating asubject having or being predisposed to any disease or disorder to whichthe subject's immune system mounts an immune response. The compositionsare useful as prophylactic vaccines, which confer resistance in asubject to subsequent exposure to infectious agents. The compositionsare also useful as therapeutic vaccines, which can be used to initiateor enhance a subject's immune response to a pre-existing antigen, suchas a tumor antigen in a subject with cancer, or a viral antigen in asubject infected with a virus. The compositions are also useful asdesensitizing vaccines, which function to “tolerize” an individual to anenvironmental antigen, such as an allergen.

The ability to target these compositions to professionalantigen-presenting cells such as dendritic cells, and the ability ofthese compositions to elicit T-cell mediated immune responses by causingcross-presentation of antigens makes these compositions especiallyuseful for eliciting a cell-mediated response to a disease-relatedantigen in order to attack the disease. Thus, in a preferred embodiment,the type of disease to be treated or prevented is a malignant tumor or achronic infectious disease caused by a bacterium, virus, protozoan,helminth, or other microbial pathogen that enters intracellularly and isattacked, i.e., by the cytotoxic T lymphocytes.

The desired outcome of a prophylactic, therapeutic or de-sensitizedimmune response may vary according to the disease, according toprinciples well known in the art. For example, an immune responseagainst an infectious agent may completely prevent colonization andreplication of an infectious agent, affecting “sterile immunity” and theabsence of any disease symptoms. However, a vaccine against infectiousagents may be considered effective if it reduces the number, severity orduration of symptoms; if it reduces the number of individuals in apopulation with symptoms; or reduces the transmission of an infectiousagent. Similarly, immune responses against cancer, allergens orinfectious agents may completely treat a disease, may alleviatesymptoms, or may be one facet in an overall therapeutic interventionagainst a disease. For example, the stimulation of an immune responseagainst a cancer may be coupled with surgical, chemotherapeutic,radiologic, hormonal and other immunologic approaches in order to affecttreatment.

i. Subjects Infected with or Exposed to Infectious Agents

Subjects with or exposed to infectious agents can be treatedtherapeutically or prophylactically with nanoparticle vaccinecompositions disclosed herein. Infectious agents include bacteria,viruses and parasites. In some instances, the subject can be treatedprophylactically, such as when there may be a risk of developing diseasefrom an infectious agent. An individual traveling to or living in anarea of endemic infectious disease may be considered to be at risk and acandidate for prophylactic vaccination against the particular infectiousagent. Preventative treatment can be applied to any number of diseaseswhere there is a known relationship between the particular disease and aparticular risk factor, such as geographical location or workenvironment.

ii. Subjects with or a Risk of Developing Malignant Tumors

In a mature animal, a balance usually is maintained between cell renewaland cell death in most organs and tissues. The various types of maturecells in the body have a given life span; as these cells die, new cellsare generated by the proliferation and differentiation of various typesof stem cells. Under normal circumstances, the production of new cellsis so regulated that the numbers of any particular type of cell remainconstant. Occasionally, though, cells arise that are no longerresponsive to normal growth-control mechanisms. These cells give rise toclones of cells that can expand to a considerable size, producing atumor or neoplasm. A tumor that is not capable of indefinite growth anddoes not invade the healthy surrounding tissue extensively is benign. Atumor that continues to grow and becomes progressively invasive ismalignant. The term cancer refers specifically to a malignant tumor. Inaddition to uncontrolled growth, malignant tumors exhibit metastasis. Inthis process, small clusters of cancerous cells dislodge from a tumor,invade the blood or lymphatic vessels, and are carried to other tissues,where they continue to proliferate. In this way a primary tumor at onesite can give rise to a secondary tumor at another site. Thecompositions and method described herein may be useful for treatingsubjects having malignant tumors.

Malignant tumors which may be treated are classified herein according tothe embryonic origin of the tissue from which the tumor is derived.Carcinomas are tumors arising from endodermal or ectodermal tissues suchas skin or the epithelial lining of internal organs and glands. Amelanoma is a type of carcinoma of the skin for which this invention isparticularly useful. Sarcomas, which arise less frequently, are derivedfrom mesodermal connective tissues such as bone, fat, and cartilage. Theleukemias and lymphomas are malignant tumors of hematopoietic cells ofthe bone marrow. Leukemias proliferate as single cells, whereaslymphomas tend to grow as tumor masses. Malignant tumors may show up atnumerous organs or tissues of the body to establish a cancer.

The types of cancer that can be treated in with the providedcompositions and methods include, but are not limited to, the following:bladder, brain, breast, cervical, colo-rectal, esophageal, kidney,liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach,uterine, and the like. Administration is not limited to the treatment ofan existing tumor or infectious disease but can also be used to preventor lower the risk of developing such diseases in an individual, i.e.,for prophylactic use. Potential candidates for prophylactic vaccinationinclude individuals with a high risk of developing cancer, i.e., with apersonal or familial history of certain types of cancer.

iii. Subjects Exposed to Allergens

The vaccine compositions may be administered to subjects for the purposeof preventing and/or attenuating allergic reactions, such as allergicreactions which lead to anaphylaxis. Allergic reactions may becharacterized by the T_(H)2 responses against an antigen leading to thepresence of IgE antibodies. Stimulation of T_(H)1 immune responses andthe production of IgG antibodies may alleviate allergic disease. Thus,the disclosed vaccine compositions may lead to the production ofantibodies that prevent and/or attenuate allergic reactions in subjectsexposed to allergens.

iv. Subjects with Immunosuppressed Conditions

Nanoparticle vaccines disclosed herein can be used for treatment ofdisease conditions characterized by immunosuppression, including, butnot limited to, AIDS or AIDS-related complex, idiopathicimmunosuppression, drug induced immunosuppression, other virally orenvironmentally-induced conditions, and certain congenital immunedeficiencies. Nanoparticle vaccine compositions can also be employed toincrease immune function that has been impaired by the use ofradiotherapy of immunosuppressive drugs (e.g., certain chemotherapeuticagents), and therefore can be particularly useful when used inconjunction with such drugs or radiotherapy.

B. Methods of Administration

In general, methods of administering vaccines are well known in the art.Any acceptable method known to one of ordinary skill in the art may beused to administer a formulation to the subject. The administration maybe localized (i.e., to a particular region, physiological system,tissue, organ, or cell type) or systemic. Vaccines can be administeredby a number of routes including, but not limited to: oral, inhalation(nasal or pulmonary), intravenous, intraperitoneal, intramuscular,transdermal, subcutaneous, topical, sublingual, or rectal means.Injections can be e.g., intravenous, intradermal, subcutaneous,intramuscular, or intraperitoneal. In some embodiments, the injectionscan be given at multiple locations.

The nanoparticle vaccines disclosed herein are particularly suitable forenteral administration. The ability to target vaccine compositions toepithelial cells in the digestive tract greatly facilitates the abilityof a vaccine to induce mucosal and systemic immunity when administeredorally. Molecules, as described above, which protect the vaccinecomposition and its constituents from hydrolysis and degradation in lowpH environments also enhance the efficacy of vaccines administeredorally.

Administration of the formulations may be accomplished by any acceptablemethod which allows an effective amount of the vaccine to reach itstarget. The particular mode selected will depend upon factors such asthe particular formulation, the severity of the state of the subjectbeing treated, and the dosage required to induce an effective immuneresponse. As generally used herein, an “effective amount” is that amountwhich is able to induce an immune response in the treated subject. Theactual effective amounts of vaccine can vary according to the specificantigen or combination thereof being utilized, the particularcomposition formulated, the mode of administration, and the age, weight,condition of the individual being vaccinated, as well as the route ofadministration and the disease or disorder.

EXAMPLES

The present invention may be further understood by reference to thefollowing non-limiting examples.

Example 1 Immune Cell-Specific Targeting of Nanoparticles

Materials and Methods:

Cells were adjusted to a concentration of 1×10⁷ cells/ml in completemedia. Plates were coated with various concentrations of anti-CDRantibodies according to established protocols. 2×10⁵ cells were platedper well. Cells were treated with 20 nM complexes either loaded orunloaded with doxorubicin and incubated at 37° C., 5% CO₂. On day 3 Tcell proliferation was analyzed with a colorimetric assay forquantification of cell proliferation and viability, WST-1®, according tomanufacturer's protocol (Roche Diagnostics GmbH, Pennsburg, Germany).

Results:

Using avidin as an adaptor element coupled to fatty acid chains whichinsert readily into PLGA particles during fabrication, biotinylatedantibodies and recombinant proteins that target different immune systemcells were attached to the surface of the particles. Thesesurface-modified particles interact specifically with cells and provideeffective delivery of anti-proliferative drugs to intracellularcompartments. For example, when modified with an antibody thatrecognizes T cells, Doxorubicin-loaded particles specifically reducedthe proliferation of those cells (FIG. 1). Similar results were shown bytargeting antigen-presenting cells using nanoparticulates presentingrecombinant T cell receptors. These data demonstrate the utility ofmodular domains for directing nanoparticles to specific subsets oftarget cells. These data indicate that this approach can be extrapolatedto target nanoparticles to other cell types, such as epithelial cellsand dendritic cells by incorporating targeting modules ontonanoparticles specific to these cell types.

Example 2 Surface Modification of Nanoparticles with Immune ModulatorsIncreases the Elicited Immune Response

Materials and Methods:

Nanoparticles were prepared by a water-oil-water emulsion method using50:50 Poly(DL-lactide-co-glycolide) from Lactel® with an inherentviscosity of 0.59 dL/g. PLGA was dissolved in methylene chloride. Forloaded particles, aqueous solutions of 10 mg chicken egg albumin(ovalbumin, OVA-antigen) was emulsified into the dissolved polymer andsonicated for 30 seconds on ice (Tekmar, Model:TMX400). The resultingwater in oil emulsion was subsequently added dropwise into thesurfactant (5% Poly(vinyl alcohol) (PVA, Sigma-Aldrich®)) and sonicatedagain for 30 seconds. This was added to a stirring 0.3% PVA solutionsurfactant solution. After 3 hours particles were centrifuged at 12,000RPM for 20 minutes and washed with DI water three times, frozen at −80°C., and lyophilized. LPS-coated particles were prepared with 20 mg/mllipopolysaccharide (Sigma®, from Escherichia coli) in the surfactant.Nanoparticles were stored after lyophilization at −20° C. Nanosphereswere characterized using scanning electron microscopy. Proteinencapsulation was quantified by dissolving the particles in DMSO for 24hr and performing a BCA Protein Assay (Pierce®).

Results:

Nanoparticulates encapsulating the model antigen, ovalbumin, weresurface modified with lipopolysaccharide (LPS) and used to induceimmunity in live animals against ovalbumin. LPS is a principal componentof the cell wall of gram-negative bacteria and is a ligand for Toll likereceptor 4, a major inducer of DC maturation and thus of T cellresponsiveness (Reis e Sousa, Semin. Immunol., 16(1):27-34 (2004);Bellou, et al., Curr. Opin. Allergy Alin. Immunol., 3(6):487-94 (2003)).This biological property of LPS was exploited to engineer an immunogenicstimulus onto nanoparticles. LPS consists of a hydrophobic fatty acidchain conjugated to hydrophilic polysaccharide chains (Mayer, Methods inMicrobiology, 18:157-207 (1985)). LPS is thus a similar composition toprotein-fatty acid conjugates and serves as a model for incorporatingprotein-fatty acid conjugates onto PLGA nanoparticles for engineeringhigh density protein display on the surface.

Only three days after immunization by subcutaneous injection, spleencells isolated from injected mice showed a remarkable memory to theinjected ovalbumin (FIG. 2A), as evidenced by proliferation of cells toimmobilized antigen in a plate. This enhanced response was not observedwith mice immunized with particles encapsulating the ovalbumin withoutLPS or with blank particles. Similar results were observed when animalswere fed LPS-modified and unmodified particles (FIG. 2B), demonstratingthe efficacy of this approach in inducing immunity by oral routes. Thisdata demonstrates that nanoparticles encapsulating antigen can be madeto be more effective vaccines by the proper choice and engineering ofrecognition elements into the surface. By derivatizing the nanoparticleswith a simple immune modulator (LPS), the nanoparticles' ability toelicit an immune response was significantly enhanced. Addition ofmodules to enhance particle targeting, internalization, endosome escape,and extracellular protection will increase the degree to which theseelements can further enhance their efficacy as vaccine vehicles.

Example 3 Endosome Disruption Enhances Antigen-Presentation by DendriticCells

Materials and Methods:

Particles prepared using the same preparation discussed in Example 2with 100 μl of endoporter added to the emulsion at a concentration of 1mg/ml.

Results:

Many pathogens make use of the acidic pH environment of endosomes andlysosomes to penetrate out from the confines of endocytic organellesinto the cytosol. Some, in fact, do this by secreting pore-formingpeptides that are low pH activated. Endoporter is a commerciallyavailable synthetic peptide that accomplishes this function (Summerton,Ann. N.Y Acad. Sci., 1058:1-14 (2005)).

Mouse bone marrow-derived dendritic cells were incubated with solubleovalbumin (0.1 mg/ml), a concentration that only inefficiently elicitsantigen presentation to CD8 T cells. Inclusion of increasingconcentrations of endoporter enhances cross presentation by 10-100-fold(depending on background) to the MHC class I-restricted,ovalbumin-specific CD8 T cell OT-I (as assayed by IL-2 release, (FIG.3)). Importantly, presentation to MHC class II-restricted CD4 T cells(OT-II) was not diminished, even after endosome disruption (FIG. 3).Similar results were obtained when the ovalbumin was targeted to thedendritic cells by conjugation to anti-DEC-205 antibody. The results ofthese experiments demonstrate the efficacy of Endoporter in enhancingthe presentation of exogenous antigens on MHC class I molecules to CD8 Tcells, presumably by enhancing their penetration into the cytosolfollowing endosomal disruption. Thus, endoporter significantly enhancesa highly inefficient but essential aspect of antigen presentationrequired for effective immunity and vaccination to pathogens. Thediagram in FIG. 5 demonstrates the mechanism for endosomal disruption byendoporter during cellular uptake of cargo.

Example 4 Co-Encapsulation of Positively Charged Macromolecules SmallMolecules, or Oligonucleotides, Enhances Endosomal Disruption byNanoparticles

Materials and Methods:

Liposomal and polymeric particles, prepared with OVA and poly(amidoamine) dendrimer generation 5 (PAMAM dendrimer G5), were incubated withbone-marrow derived dendritic cells (BMDCs) or bone marrow derivedmacrophages (BMDMs) for 24 hours. Endosomal disruption was measuredindirectly through assessing the level of cross-presentation by thecells. Cross-presentation is a process where antigen escapes theendosomal compartment and is presented by the MHC in the cytosol. Thispresentation can be measured with an antibody (25.D16-PE). The amount of25.D16-PE-positive cells represents the degree of cross-presentationmediated by endosomal disruption.

In another preparation, the PLGA nanoparticles were prepared carryingcarbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) via adendrimer (G4) or cyclodextrin.

In another preparation, the monophosphoryl lipid A (MPLA) nanoparticleswere prepared carrying G5 and/or CpG.

The prepared nanoparticles were tested for their ability to disruptendosomes. Particles co-encapsulating the OVA peptide or GFP associatedwith the dendrimer were incubated at a concentration of 50 μg/ml with1E5 BMDCs for 24 hours. Liposomal disruption was assessed bothqualitatively via fluorescence microscopy of GFP in the cytosol orquantitatively by amount of OVA antigen presented via MHC Class I.Antigen presentation on Class I MHC takes place via cytosolic transportand OVA peptide/MHC Class I was detected specifically using the antibody24D16-PE. FIGS. 6-7 show amount of antibody bound to the surface of DCsafter particle internalization and compared to PLGA nanoparticlesencapsulating the same antigen. FIG. 8 shows endosomal disruptionassessed by quantitating cytosolic fluorescence in the presence andabsence of a trifluoromethoxy phenylhydrazone (FCCP) a protonophore anduncoupler of oxidative phosphorylation in the mitochondria. FCCP ishosted in the dendrimer via a conjugated cyclodextrin moleculeaccommodating the hydrophobic FCCP molecule. The dendrimer/Cyclodextrinratio is 1:5 and the figure shows that FCCP hosting can take place evenwithout cyclodextrin attached since FCCP maybe associated with thedendrimer cavity itself.

It is therefore expected that FCCP would further disrupt theendosomal/lysosomal compartment resulting in increased fluorescencecompared to dendrimer alone or the drug alone as shown in FIG. 7.

Results:

FIGS. 6 and 7 demonstrate that dendrimer (G5) encapsulation with antigenOVA increases cross-presentation in liposomal and polymericnanoparticles (PLGA or PLGA-PEG nanoparticles) when co-incubated withmouse BMDCs or BMDMs.

FIG. 8 demonstrates that incorporation of FCCP significantly enhancesthe ability of PLGA-G4 or PLGA-G4-cyclodextrin nanoparticles to disruptendosomes.

FIG. 9 demonstrates that the highest percentage of transfection of bonemarrow derived dendritic cells with GFP occurred when the cells weretransfected with MPLA nanoparticles co-encapsulating GFP, the dendrimerG5 and CpG. The graph also describes the importance of having both TLRligands associated with the same particle in that MPLA/G5 LED &−/(G5+CpG) LED is not as efficient in transfection compared toMPLA/(G5+CpG).

Example 5 Surface Modification of Nanoparticles with Protective CoatingsDecreases Particle Degradation and Antigen Release

Materials and Methods:

Particles were prepared as in Example 2. After lyophilization,biotin-elastin was prepared by biotinylation with NHS-LC-biotin (PierceChemicals). NHS-LC-biotin was incubated with 10 mg of particles (roomtemperature, 1 hour) at a concentration of 20 mg/ml. Followingincubation, particles were washed by centrifugation 3× with deionizedwater and freeze-dried for further use. Controlled release studies wereperformed at the indicated pH.

Results:

One advantage of antigen delivery using particles is the possibility ofprotecting the antigen against destruction in the GI tract followingoral delivery. Elastin and poly(methacrylic) acid (PMAA) were conjugatedto nanoparticles. As pH responsive polymers, both are in extendedconfigurations at pH 7.4 and shrink rapidly (within seconds) uponexposure to lower pH environments (below pH 5 for elastin) and (below pH5-6 for poly(methacrylic acid)). These data indicate that the additionof a pH responsive polypeptide or polymer to the surface of thenanoparticles can impart a protective effect to the particle affectingits degradation rate and antigen release (FIG. 4). The mechanism behindthis protective ability may be due to the aggregation of the polymer atlower pH, restricting water entry and reducing the hydrolysis of theparticle. Polymers such as PMAA in conjunction with poly(ethyleneglycol) (PEG) have been used in past applications for enhancing the oraldelivery of chemotherapeutic drugs, and thus there is good precedencefor the use of these polymers as ‘shielding’ components in oral delivery(Blanchette and Peppas, Ann. Biomed. Eng., 33(2):142-9 (2005);Blanchette and Peppas, J. Biomed. Mater. Res. A, 72(4):381-8 (2005)).

Example 6 Surface Modification of Nanoparticles Targets DifferentDendritic Cell Subsets

Materials and Methods:

Generation of Peptide-Loaded Nanoparticles (NPs)

PLGA NPs containing avidin on the surface were prepared using methodsdescribed by Park (Park et al., J. Control. Release, 156:109-115, 2011)and Fahmy (Fahmy et al., Biomaterials, 26:5727-5736, 2005). The preparedNPs included blank NP (no peptide), coumarin-labeled blank NP(NP-coumarin), NP-influenza-matrix peptide (FMP) (incorporating HLA A2.1FMP sequence GILGFVFTL), NP-CEF (incorporating CEF pool peptide, pool of32 peptides from EBV, CMV, and influenza virus, Anaspec), and NP-SOX2(22 15-mer SOX2 peptides; Table 1). The amount of each peptide in theNPs was as follows: NP-FMP (9 μg/mg NP), NP-CEF (0.56 μg/mg NP), andNP-SOX2 (4.1 μg/mg NP).

TABLE 1 A list of SOX2 overlapping peptidesequences loaded in nanoparticles. Serial SOX2 Peptide Number SequenceSEQ ID NO. 1 LGAEWKLLSETEKR SEQ ID NO: 1 2 EWKLLSETEKRPFI SEQ ID NO: 2 3LLSETEKRPFIDEAK SEQ ID NO: 3 4 TEKRPFIDEAKRLRA SEQ ID NO: 4 5PFIDEAKRLRALHMK SEQ ID NO: 5 6 EAKRLRALHMKEH SEQ ID NO: 6 7KRLRALHMKEHPDYK SEQ ID NO: 7 8 ALHMKEHPDYKYRPR SEQ ID NO: 8 9KEHPDYKYRPRRKTK SEQ ID NO: 9 10 DYKYRPRRKTKTLMK SEQ ID NO: 10 11RPRRKTKTLMKKDKY SEQ ID NO: 11 12 KTKTLMKKDKYTLPG SEQ ID NO: 12 13LMKKDKYTLPGGLLA SEQ ID NO: 13 14 DKYTLPGGLLAPGG SEQ ID NO: 14 15TLPGGLLAPGGNSMA SEQ ID NO: 15 16 GLLAPGGNSMASGVG SEQ ID NO: 16 17PGGNSMASGVGVGAG SEQ ID NO: 17 18 SMASGVGVGAGLGAG SEQ ID NO: 18 19GVGVGAGLGAGVNQR SEQ ID NO: 19 20 GAGLGAGVNQRMDSY SEQ ID NO: 20 21GAGVNQRMDSYAHM SEQ ID NO: 21 22 VNQRMDSYAHMNGWS SEQ ID NO: 22

TLR and/or antibody (Ab)-coated NPs were prepared by adding biotinylatedLPS (InvivoGen), polyinosinic-polycytidylic acid [poly (I:C)](InvivoGen), BDCA3 Ab (Miltenyi Biotec), or DC-SIGN Ab (Miltenyi Biotec)at the concentration of 5 μg Ab per milligram of NPs. The vials weregently rotated for 15 min. They were then centrifuged at 1200 rpm for 5min to remove the supernatant and washed twice to remove any solubleligand prior to use in experiments.

NP Targeting Experiments

Coumarin-labeled NPs were coated with either anti-BDCA3 or anti DC-SIGNantibody and co-cultured with peripheral blood mononuclear cells (PBMCs)for 30 min at 4° C.

Results:

FIG. 10 demonstrates specific targeting of BDCA3+ and DC-SIGN+ dendriticcells with targeted NPs. The change in MFI of coumarin in targeted APCsover that in non-targeted APCs was greatest when BDCA3-targeted NPs wereincubated with BDCA3+ myeloid dendritic cells (MDCs) and whenDC-SIGN-targeted NPs were incubated with DC-SIGN+DCs.

Example 7 Targeting the Influenza Antigen to Dendritic Cells Potentiatesthe Immune Response

Generation of Peptide-Loaded NPs

PLGA NPs containing NP-influenza-matrix peptide (FMP) (incorporating HLAA2.1 FMP sequence GILGFVFTL) were prepared as described in Example 6.

Generation of DCs

Monocyte-derived DCs (Mo-DCs) were generated from PBMCs, as described byDhodapkar (Dhodapkar et al., Proc. Natl. Acad. Sci. USA, 102:2910:2915,2005). Briefly, CD14+ monocytes were isolated from PBMCs byimmunomagnetic bead selection using CD14 beads following themanufacturer's protocol (Miltenyi Biotec). CD14+ cells were suspended in1% healthy donor plasma in RPMI 1640 (Cellgro), supplemented with IL-4(25 μg/ml; R&D Systems) and GM-CSF (20 ng/ml sargramostim (Leukine);Genzyme) on days 0, 2, and 4 of culture. Immature Mo-DCs were harvestedon days 5-6 and used for the experiments described below. The CD14−fraction of PBMCs was cultured in the presence of 5% pooled human serum(Labquip) in RPMI 1640. BDCA3+ MDCs were isolated from the PBMCs usingBDCA3 MACS beads (Miltenyi Biotec).

NP Uptake Experiments

The effects of NPs on isolated BDCA3+ MDCs and DC-SIGN+ Mo-DCs werestudied by incubating the cells overnight with NP-FMP (peptideconcentration 0.05 μg/ml) at 37° C. BDCA3+ MDCs isolated from healthydonor buffy coats or DC-SIGN+ Mo-DC were loaded with NP-FMP at 37° C.and supernatants were collected after 24 h. Cytokines were quantifiedusing VeriPlex Human Cytokine ELISA (PBL IFN source) and data wereanalyzed by Q-View 2.160 software (Quansys Biosciences). BDCA3+ MDCs andDC-SIGN DCs incubated alone were used as negative controls.

Results:

FIGS. 11A-11D demonstrate that targeting the influenza antigen (FMP) toBDCA3+ or DC-SIGN+DC subsets potentiates the immune response. The graphshows mean cytokine expression levels (IL-15, IFN-λ and TNF-α in pg/ml,and IL-6 and IL-8 in pg/dl, ±SEM) per 30,000 APCs for cells obtainedfrom three different healthy donors. *p<0.05.

Example 8 Delivery of Combination of Peptides by Dendritic CellsStimulates a Specific and Multivalent T Cell Response

Materials and Methods

Generation of CEF Peptide-Loaded NPs

Nanoparticles loaded with CEF combination of 32 peptides were preparedas described in Example 6.

Antigen-Specific T Cell Stimulation

Day 6 immature Mo-DCs or freshly isolated BDCA3+ MDCs were loaded witheither blank NPs or NPs encapsulated with CEF pool peptide (NP-CEF) for1 h. After overnight culture in 1% plasma, NP-loaded DCs were used tostimulate autologous T cells at a DC/T cell ratio of 1:30 in thepresence of IL-2 (10 μg/ml at days 4 and 7; Chiron). After 10-12 days inculture, flow cytometry was performed to detect the presence ofantigen-specific T cells intracellular cytokine secretion assay forIFN-γ, with the peptides used for initial T cell stimulation in thepresence of anti-CD28 and anti-CD49D (1 μg/ml).

Results:

BDCA3+ MDCs were able to stimulate Ag-specific IFN-γ-secreting T cellsin response to NP-CEF. Importantly, the elicited immune responseincluded reactivities against multiple peptides within the mix.Induction of IFN-γ production by CD8 lymphocytes from a healthy donor inresponse to antigen-specific stimulation by NP-CEF-loaded autologousBDCA3+ MDCs and absence of stimulation by autologous BDCA3+ MDCs loadedwith blank NP FIG. 12 demonstrates the mean percentage (%, ±SEM) of CD8cells producing IFN-γ when stimulated by Mo-DCs (n=8) or BDCA3+ MDCs(n=3) loaded with either blank NP or NP-CEF. *p=6×10⁻⁴ (blank NP versusNP-CEF for Mo-DCs) and p=4.9×10⁻² (blank NP versus NP-CEF forBDCA3+MDCs).

The multivalent nature of the CD8+ T cell response following stimulationby Mo-DCs or BDCA3+ MDCs loaded with either blank NP or NP-CEF wasconfirmed when the CD8+ T cells were re-stimulated with individualpeptide components (Pep1-Pep18; described in Table 2) of the CEF peptidepool. Blank NP and NP-CEF restimulated with CEF pool peptide were usedas negative and positive controls, respectively.

TABLE 2 A list of combinations of CEFpeptides from EBV, HCMV and influenza. Pep- HLA Protein & Peptide SEQ IDtide Allele Virus Region Sequence NO. Pep 1 A1 INFLU- PB1 VSDGGPNLYSEQ ID ENZA A (591-599) NO: 23 Pep 2 A2 EBV BMLF1 GLCTLVAML SEQ ID(259-267) NO: 24 Pep 3 A2 INFLU- MATRIX 1 GILGFVFTL SEQ ID ENZA A(58-66) NO: 25 Pep 4 A3 INFLU- NP ILRGSVAHK SEQ ID ENZA A (265-273)NO: 26 Pep 5 A3 EBV BRLF1 RVRAYTYSK SEQ ID (148-156) NO: 27 Pep 6 A3 EBVEBNA 3A RLRAEAQVK SEQ ID (603-611) NO: 28 Pep 7 A11 EBV EBNA 3BIVTDFSVIK SEQ ID (416-424) NO: 29 Pep 8 A11 EBV BRLF1 ATIGTAMYK SEQ ID(134-143) NO: 30 Pep 9 A24 EBV BRLF1 DYCNVLNKEF SEQ ID (28-37) NO: 31Pep 10 A68 INFLU- NP KTGGPIYKR SEQ ID ENZA A (91-99) NO: 32 Pep 11 B7EBV EBNA 3A RPPIFIRRL SEQ ID (379-387) NO: 33 Pep 12 B8 EBV EBNA 3AQAKWRLQTL SEQ ID (158-166) NO: 34 Pep 13 B8 EBV EBNA 3A FLRGRAYGL SEQ ID(325-333) NO: 35 Pep 14 B8 EBV BZLF1 RAKFKQLL SEQ ID (190-197) NO: 36Pep 15 B27 EBV EBNA 3C RRIYDLIEL SEQ ID (258-266) NO: 37 Pep 16 B27INFLU- NP SRYWAIRTR SEQ ID ENZA A (383-391) NO: 38 Pep 17 B35 EBVEBNA 3A YPLHEQHGM SEQ ID (458-466) NO: 39 Pep 18 B44 HCMV Pp65EFFWDANDIY SEQ ID (512-521) NO: 40

Example 9 Delivery of Combinations of Tumor Antigen Peptides of SOX2 toDendritic Cells Leads to Stimulation of Antigen-Specific CD4 as Well asCD8 T Cells

Materials and Methods:

Generation of CEF Peptide-Loaded NPs

Nanoparticles loaded with tumor antigen peptides of SOX2 were preparedas described in Example 6.

Antigen-Specific T Cell Stimulation

Day 6 immature Mo-DCs were loaded with either blank NPs or NPsencapsulated with SOX2 pool peptide (NP-SOX2, Table 1) for 4 hours.After overnight culture in 1% plasma, NP-loaded DCs were used tostimulate autologous T cells at a DC/T cell ratio of 1:30 in thepresence of IL-2 (10 μg/ml at days 4 and 7; Chiron). After 10-12 days inculture, flow cytometry was performed by intracellular cytokinesecretion assay for IFN-γ, with the peptides used for initial T cellstimulation in the presence of anti-CD28 and anti-CD49D (1 μg/ml).

T cells were restimulated with NP-SOX2 loaded DCs on days 7 and 14 inthe presence of IL-2 (10 μg/ml) as well as IL-7 and IL-15 (both at 5μg/ml; R&D Systems). For some experiments, DCs were matured overnightwith LPS (50 ng/ml; Sigma-Aldrich) or poly(I:C) (25 μg/ml;Sigma-Aldrich) or cytokine mixture [IL-6 (0.01 μg/ml; R&D Systems),IL-1β (0.01 μg/ml; R&D Systems), TNF-α (0.01 μg/ml; R&D Systems), andPGE2 (1 μg/ml, Sigma-Aldrich)] after loading with NPs.

Results:

NP-SOX2-loaded autologous DCs were used to stimulate T cells becauseSOX2 peptides are 15 aa long and require active processing for antigenpresentation. DCs loaded with NP-SOX2 were able to stimulate bothSOX2-specific CD4 and CD8+ T cells in culture. Taken together these datademonstrate that both BDCA3+ and Mo-DC-SIGN+NP-loaded DCs are equallyeffective at generating antigen-specific human T cells in culture,including against complex peptide mixtures from viral and tumor antigensacross multiple MHC molecules.

It is understood that the disclosed invention is not limited to theparticular methodology, protocols, and reagents described as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

We claim:
 1. A vaccine composition for inducing an immune response comprising one or more antigens encapsulated in or incorporated into a polymeric nanoparticle, adaptor elements bound to or incorporated in the nanoparticles, and one or more functional elements selected from the group consisting of adjuvant or immune potentiators, molecular recognition factors, transport mediation elements, and intracellular uptake mediators, wherein the antigen or functional elements are bound to the nanoparticles by adaptor elements.
 2. The composition of claim 1 wherein the antigen is selected from the group consisting of viral, bacterial, parasitic, allergen, toxoid, tumor-specific and tumor-associated antigens.
 3. The composition of claim 1 wherein the adaptor elements are coupled directly to functional elements by covalent bonds.
 4. The composition of claim 1 wherein the adaptor element is conjugated to an affinity tag.
 5. The composition of claim 4 wherein the adaptor element is coupled to a functional element by the non-covalent interaction of the affinity tag conjugated to the adaptor element and a complementary affinity tag conjugated to the functional element.
 6. The composition of claim 1 wherein the functional element is selected from the group consisting of dendritic cell targeting molecules, epithelial cell targeting molecules, pH-sensitive or non-pH-sensitive molecules which protect the vaccine composition from hydrolysis and degradation in low pH environments, and endosome-disrupting agents.
 7. The composition of claim 1 further comprising materials selected from the group consisting of adjuvants, immune modulators, contrast agents and other markers.
 8. The composition of claim 7 wherein the adjuvant or immune modulator is selected from the group consisting of a cytokine, an interleukin, an interferon, a macrophage colony stimulating factor, a tumor necrosis factor, and a member of the B7 family of co-stimulatory molecules.
 9. The composition of claim 7 wherein the adjuvant or immune modulator is a glycolipid that is a stimulator of natural killer T cell-mediated immune responses.
 10. The composition of claim 9 wherein the glycolipid is α-galactosylceramide.
 11. The composition of claim 1 wherein the adaptor elements comprise elements selected from the group consisting of fatty acids, hydrophobic or amphipathic peptides, and hydrophobic polymers.
 12. The composition of claim 4 wherein the affinity tag comprises avidin or streptavidin.
 13. The composition of claim 1 wherein the functional elements are targeting molecules that target the particles to a cell-type specific receptor.
 14. The composition of claim 13 wherein the targeting molecules are selected from the group consisting of monoclonal or polyclonal antibodies, targeting molecules that target the composition to a receptor on dendritic cells, dendritic cell receptors, and targeting molecules which target the composition to a receptor on epithelial cells.
 15. The composition of claim 1 wherein the functional elements further comprise molecules which protect the composition from hydrolysis and degradation in low-pH environments selected from the group consisting of elastin, poly(methacrylic acid), and poly(ethylene) glycol.
 16. The composition of claim 1 further comprising an endosome-disrupting agent encapsulated in the polymeric nanoparticle.
 17. The composition of claim 1 further comprising a contrast agent, a fluorescent tag, or a radionuclide.
 18. The composition of claim 1 further comprising pharmaceutically acceptable excipients suitable for enteral administration.
 19. The composition of claim 1 further comprising pharmaceutically acceptable excipients suitable for parenteral administration.
 20. A method for inducing an immune response to an antigen comprising administering to a subject in need an effective amount of any of the compositions of claim
 1. 21. The method of claim 20 wherein the composition is administered enterally.
 22. The method of claim 20 wherein the composition is administered parenterally.
 23. A method for inducing an immune response to multiple antigens comprising administering to a subject in need an effective amount of the composition of claim
 1. 24. The method of claim 23 wherein the composition is administered enterally.
 25. The method of claim 24 wherein the composition is administered parenterally. 